Methods of treating elevated plasma cholesterol

ABSTRACT

The present invention relates to compounds of Formula I, pharmaceutically acceptable salts, solvates or formulations thereof. Compounds of Formula I increase significantly low density lipoprotein receptor and are useful for preventing and treating of elevated cholesterol.

FIELD OF THE INVENTION

The present invention is directed to pyridoxine derived compounds ofFormula I, pharmaceutically acceptable salts or solvates thereof,pharmaceutical formulations including one or more compounds of FormulaI, methods synthesizing or manufacturing a compound of Formula 1, anduse the compounds as modulators (e.g., enhancers) of low-densitylipoprotein receptor (LDLR). Compounds of the present invention areuseful for prophylaxis, treatment of elevated plasma cholesterol, delayin the onset, or delay in the progression of atheroscleroticcardiovascular diseases (ASCVD).

BACKGROUND OF THE INVENTION

A disease of the arteries, atherosclerosis, is recognized to be theleading cause of death in Europe, Canada, and the United States. Thesequence of events leading to atherosclerosis and occlusive heartdisease pathology is well known. The earliest stage in this sequence isthe formation of lesions, “fatty streaks,” in the major blood vessels.It is postulated that most of the cholesterol found within the fattystreaks, in turn, gives rise to development of the “fibrous plaque,”cells plus matrix protein form a fibrous cap that covers a deeperdeposit of cell debris and extracellular lipids. Epidemiologicalinvestigation has firmly established hyperlipidemia (i.e., high plasmalipids) as a primary risk factor in causing cardiovascular disease (CVD)due to atherosclerosis. The most important factor leading toatherogenesis is longstanding hypercholesterolemia, high circulatinglevels of low-density lipoprotein (LDL) that result in cholesteroldepositions in arterial vessels.

Leaders of the medical profession have placed renewed emphasis recentlyon lowering plasma cholesterol levels, and LDL-cholesterol, as anessential step in prevention of ASCVD. Western populations are at a highrisk. Additional risk factors include: being of the male sex,postmenopausal women, hypertension, glucose intolerance, insulinresistance, tobacco use, physical inactivity, stress, and leftventricular hypertrophy. Successful treatment of hyperlipidemia in thegeneral population, and in diabetic subjects, is of exceptional medicalimportance.

Plasma LDL particles, which carry the majority of total circulatingcholesterol, are cleared by binding to hepatic LDL receptors (LDLR),being endocytosed, and being catabolized.

SUMMARY OF THE INVENTION

The compounds of the present invention are useful for enhancinglow-density lipoprotein receptor (LDLR) activity and, in particular, forinducing low-density lipoprotein receptor production, and for treatingelevated plasma cholesterol and related conditions. The presentinvention relates to a series of low-density lipoprotein receptorinducers derived from pyridoxine and pharmaceutically acceptablederivatives thereof (e.g., salts and solvates) described in U.S. Pat.No. 8,742,123 B2, which is hereby incorporated by reference.

The compounds of the present invention are useful for enhancing LDLRactivity and, in particular, for inducing LDLR production, and fortreating elevated plasma cholesterol and related conditions. The presentinvention relates to a series of LDLR inducers derived from pyridoxineand pharmaceutically acceptable derivatives thereof (e.g., salts andsolvates) described in U.S. Pat. No. 8,742,123, which is herebyincorporated by reference.

Accordingly, in the first aspect, the invention features a method oflowering low-density lipoprotein (LDL)-cholesterol level in thebloodstream of a subject. This method includes administering to thesubject a LDL-lowering amount of a compound having the formula:

where Y—X is —C(R₇)(R_(7a))N(R′)C(O)—; —C(R₇)(R_(7a))OC(O)—;—C(R₇)(R_(7a))N(R′)C(R₆)(R_(6a))—; —C(R₇)(R_(7a))S(O)₂C(R₆)(R_(6a))—;—S(O)₂C(R₆)(R_(6a))—; —C(R₇)(R_(7a))C(R₆)(R_(6a))—; —O—C(R₆)(R_(6a))—;—N(R′)C(R₆)(R_(6a))—; or —C(R₇)(R_(7a))OC(R₆)(R_(6a))—, where each ofR₆, R_(6a), R₇, and R_(7a), is, independently, selected from H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle, R′ is selectedfrom H, C₁₋₆ alkyl, benzyl, S(O)₂R″, and C(O)R″, and R″ is selected fromC₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle; when Q isH, P and R₃ are absent; when Q is CH₃, P and R₃ are absent; Q is CH₂, orC(O); when P is H, R₃ is absent; P is —O—, —N(R₈)(R_(8a)), or is absent,where the R₈ and R_(8a) are selected independently from H, C₁₋₆ alkyl,C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle; R₁ is absent, C₁₋₆alkyl, C₁₋₆ branched alkyl, C₁₋₆ alkene, halogen (F, Cl, Br, I), OH,—O—(C₁₋₆ alkyl), —O—(C₁₋₆ branched alkyl), —C(O)(R₉), —C(O)O(R₉),—C(O)N(R₉)(R_(9a)), or —S(O)₂N(R₉)(R_(9a)), where R₉ and R_(9a) areselected independently from H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, and heterocycle; R₂ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl,C₁₋₆ alkene, halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆branched alkyl), —C(O)(R₁₀), —C(O)O(R₁₀), or —C(O)N(R₁₀)(R_(10a)), whereR₁₀ and R_(10a) are selected independently from H, C₁₋₆ alkyl, C₁₋₆fluoroalkyl, benzyl, phenyl, and heterocycle; or R₁ and R₂ are orthosubstituents that together form a carbocyclic or heterocyclic ringsystem; R₃ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl,heterocycle, or is absent; R₅ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl,benzyl, phenyl, or heterocycle; or R₃ and R₅ combine to form aheterocyclic ring system; R₄ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, or heterocycle; and m is 0 or 1; or a pharmaceuticallyacceptable salt or solvate thereof.

In some embodiments of the first aspect of the invention, Q is CH₂, P is—O—, R₃ is H, Y—X is —CH₂NHCH₂— or —CH₂NHC(O)—, R₁ is selected from ahalogen, —OH or —OCH₃, R₂ is absent or selected from —OH and a halogen,R₄ is H or benzyl, and R₅ is H or benzyl, or a pharmaceuticallyacceptable salt or solvate thereof.

In other embodiments of the first aspect of the invention, Q is CH₂, Pis —O—, R₃ is CH₃, Y—X is —CH₂OCH₂—, R₁ is selected from a halogen, —OHor —OCH₃, R₂ is absent or selected from —OH and a halogen, R₄ is H orbenzyl, and R₅ is H or benzyl or a pharmaceutically acceptable salt orsolvate thereof.

In an embodiment of the first aspect of the invention, the compound is5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In one embodiment,5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g.,atorvastatin).

In another embodiment of the first aspect of the invention, the compoundis5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In one embodiment,5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g.,atorvastatin).

In a further embodiment of the first aspect of the invention, thecompound is5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In one embodiment,5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g.,atorvastatin).

In additional embodiments of the first aspect of the invention, thesubject has been diagnosed with atherosclerosis, hypercholesterolemia,hypertriglyceridemia, diabetic complications, dyslipidemia,hyperlipidemia, hypoalphalipoproteinemia, metabolic syndrome, stroke,vascular dementia, chronic kidney disease, coronary heart disease,coronary artery disease, retinopathy, inflammation, thrombosis,peripheral vascular disease or congestive heart failure. In someembodiments, the hypercholesterolemia is heterozygous familialhypercholesterolemia, and in other embodiments, the hypercholesterolemiais homozygous familial hypercholesterolemia.

In some embodiments of the first aspect of the invention, the subjecthas been diagnosed with atherosclerosis. In some embodiments of thefirst aspect of the invention, the subject has been diagnosed withhypercholesterolemia. In some embodiments of the first aspect of theinvention, the subject has been diagnosed with hypertriglyceridemia. Insome embodiments of the first aspect of the invention, the subject hasbeen diagnosed with diabetic complications. In some embodiments of thefirst aspect of the invention, the subject has been diagnosed withdyslipidemia. In some embodiments of the first aspect of the invention,the subject has been diagnosed with hyperlipidemia. In some embodimentsof the first aspect of the invention, the subject has been diagnosedwith hypoalphalipoproteinemia. In some embodiments of the first aspectof the invention, the subject has been diagnosed with metabolicsyndrome. In some embodiments of the first aspect of the invention, thesubject has been diagnosed with stroke. In some embodiments of the firstaspect of the invention, the subject has been diagnosed with vasculardementia. In some embodiments of the first aspect of the invention, thesubject has been diagnosed with chronic kidney disease. In someembodiments of the first aspect of the invention, the subject has beendiagnosed with coronary heart disease. In some embodiments of the firstaspect of the invention, the subject has been diagnosed with coronaryartery disease. In some embodiments of the first aspect of theinvention, the subject has been diagnosed with retinopathy. In someembodiments of the first aspect of the invention, the subject has beendiagnosed with inflammation. In some embodiments of the first aspect ofthe invention, the subject has been diagnosed with thrombosis. In someembodiments of the first aspect of the invention, the subject has beendiagnosed with peripheral vascular disease. In some embodiments of thefirst aspect of the invention, the subject has been diagnosed withcongestive heart failure

In a second aspect, the invention features a method of treatingatherosclerosis, hypercholesterolemia, hypertriglyceridemia, diabeticcomplications, dyslipidemia, hyperlipidemia, hypoalphalipoproteinemia,metabolic syndrome, stroke, vascular dementia, chronic kidney disease,coronary heart disease, coronary artery disease, retinopathy,inflammation, thrombosis, peripheral vascular disease or congestiveheart failure. This method includes administering to a subject in needthereof, a therapeutically effective amount of a compound having theformula:

where Y—X is —C(R₇)(R_(7a))N(R′)C(O)—; —C(R₇)(R_(7a))OC(O)—;—C(R₇)(R_(7a))N(R′)C(R₆)(R_(6a))—; —C(R₇)(R_(7a))S(O)₂C(R₆)(R_(6a))—;—S(O)₂C(R₆)(R_(6a))—; —C(R₇)(R_(7a))C(R₆)(R_(6a))—; —O—C(R₆)(R_(6a))—;—N(R′)C(R₆)(R_(6a))—; or —C(R₇)(R_(7a))OC(R₆)(R_(6a))—, where each ofR₆, R_(6a), R₇, and R_(7a), is, independently, selected from H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle, R′ is selectedfrom H, C₁₋₆ alkyl, benzyl, S(O)₂R″, and C(O)R″, and R″ is selected fromC₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle; when Q isH, P and R₃ are absent; when Q is CH₃, P and R₃ are absent; Q is CH₂, orC(O); when P is H, R₃ is absent; P is —O—, —N(R₈)(R_(8a)), or is absent,where R₈ and R_(8a) are selected independently from H, C₁₋₆ alkyl, C₁₋₆fluoroalkyl, benzyl, phenyl, and heterocycle; R₁ is absent, C₁₋₆ alkyl,C₁₋₆ branched alkyl, C₁₋₆ alkene, halogen (F, Cl, Br, I), OH, —O—(C₁₋₆alkyl), —O—(C₁₋₆ branched alkyl), —C(O)(R₉), —C(O)O(R₉),—C(O)N(R₉)(R_(9a)), or —S(O)₂N(R₉)(R_(9a)), where R₉ and R_(9a) areselected independently from H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, and heterocycle; R₂ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl,C₁₋₆ alkene, halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆branched alkyl), —C(O)(R₁₀), —C(O)O(R₁₀), or —C(O)N(R₁₀)(R_(10a)), whereR₁₀ and R_(10a) are selected independently from H, C₁₋₆ alkyl, C₁₋₆fluoroalkyl, benzyl, phenyl, and heterocycle; or R₁ and R₂ are orthosubstituents that together form a carbocyclic or heterocyclic ringsystem; R₃ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl,heterocycle, or is absent; R₅ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl,benzyl, phenyl, or heterocycle; or R₃ and R₅ combine to form aheterocyclic ring system; R₄ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, or heterocycle; and m is 0 or 1; or a pharmaceuticallyacceptable salt or solvate thereof.

In some embodiments of the second aspect of the invention, the methodtreats atherosclerosis. In some embodiments of the second aspect of theinvention, the method treats hypercholesterolemia. In some embodimentsof the second aspect of the invention, the method treatshypertriglyceridemia. In some embodiments of the second aspect of theinvention, the method treats diabetic complications. In some embodimentsof the second aspect of the invention, the method treats dyslipidemia.In some embodiments of the second aspect of the invention, the methodtreats hyperlipidemia. In some embodiments of the second aspect of theinvention, the method treats hypoalphalipoproteinemia. In someembodiments of the second aspect of the invention, the method treatsmetabolic syndrome. In some embodiments of the second aspect of theinvention, the method treats stroke. In some embodiments of the secondaspect of the invention, the method treats vascular dementia. In someembodiments of the second aspect of the invention, the method treatschronic kidney disease. In some embodiments of the second aspect of theinvention, the method treats coronary heart disease. In some embodimentsof the second aspect of the invention, the method treats coronary arterydisease. In some embodiments of the second aspect of the invention, themethod treats retinopathy. In some embodiments of the second aspect ofthe invention, the method treats inflammation. In some embodiments ofthe second aspect of the invention, the method treats thrombosis. Insome embodiments of the second aspect of the invention, the methodtreats peripheral vascular disease. In some embodiments of the secondaspect of the invention, the method treats congestive heart failure.

In some embodiments of the second aspect of the invention, Q is CH₂, Pis —O—, R₃ is H, Y—X is —CH₂NHCH₂— or —CH₂NHC(O)—, R₁ is selected from ahalogen, —OH or —OCH₃, R₂ is absent or selected from —OH and a halogen,R₄ is H or benzyl, and R₅ is H or benzyl, or a pharmaceuticallyacceptable salt or solvate thereof.

In other embodiments of the second aspect of the invention, Q is CH₂, Pis —O—, R₃ is CH₃, Y—X is —CH₂OCH₂—, R₁ is selected from a halogen, —OHor —OCH₃, R₂ is absent or selected from —OH and a halogen, R₄ is H orbenzyl, and R₅ is H or benzyl or a pharmaceutically acceptable salt orsolvate thereof.

In a particular embodiment of the second aspect of the invention, thecompound is5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In one embodiment,5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g.,atorvastatin).

In another embodiment of the second aspect of the invention, thecompound is5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In one embodiment,5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g.,atorvastatin).

In a further embodiment of the second aspect of the invention, thecompound is5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In one embodiment,5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g.,atorvastatin).

In an embodiment of the second aspect of the invention, thehypercholesterolemia is heterozygous familial hypercholesterolemia, andin another embodiment of the second aspect of the invention, thehypercholesterolemia is homozygous familial hypercholesterolemia.

In additional embodiments of the first aspect or the second aspect ofthe invention, a pharmaceutical composition including the compound orthe salt or solvate thereof is administered to the subject.

In a third aspect, the invention features a method of inducing lowdensity lipoprotein receptor (LDLR) expression in a cell. This methodincludes contacting the cell with an effective amount of a compoundhaving the formula:

where Y—X is —C(R₇)(R_(7a))N(R′)O(O)—; —C(R₇)(R_(7a))OC(O)—;—C(R₇)(R_(7a))N(R′)C(R₆)(R_(6a))—; —C(R₇)(R_(7a))S(O)₂C(R₆)(R_(6a))—;—S(O)₂C(R₆)(R_(6a))—; —C(R₇)(R_(7a))C(R₆)(R_(6a))—; —O—C(R₆)(R_(6a))—;—N(R′)C(R₆)(R_(6a))—; or —C(R₇)(R_(7a))OC(R₆)(R_(6a))—, where each ofR₆, R_(6a), R₇, and R_(7a), is, independently, selected from H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle, R′ is selectedfrom H, C₁₋₆ alkyl, benzyl, S(O)₂R″, and C(O)R″, and R″ is selected fromC₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle; when Q isH, P and R₃ are absent; when Q is CH₃, P and R₃ are absent; Q is CH₂, orC(O); when P is H, R₃ is absent; P is —O—, —N(R₈)(R_(8a)), or is absent,where R₈ and R_(8a) are selected independently from H, C₁₋₆ alkyl, C₁₋₆fluoroalkyl, benzyl, phenyl, and heterocycle; R₁ is absent, C₁₋₆ alkyl,C₁₋₆ branched alkyl, C₁₋₆ alkene, halogen (F, Cl, Br, I), OH, —O—(C₁₋₆alkyl), —O—(C₁₋₆ branched alkyl), —C(O)(R₉), —C(O)O(R₉),—C(O)N(R₉)(R_(9a)), or —S(O)₂N(R₉)(R_(9a)), where R₉ and R_(9a) areselected independently from H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, and heterocycle; R₂ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl,C₁₋₆ alkene, halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆branched alkyl), —C(O)(R₁₀), —C(O)O(R₁₀), or —C(O)N(R₁₀)(R_(10a)), whereR₁₀ and R_(10a) are selected independently from H, C₁₋₆ alkyl, C₁₋₆fluoroalkyl, benzyl, phenyl, and heterocycle; or R₁ and R₂ are orthosubstituents that together form a carbocyclic or heterocyclic ringsystem; R₃ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl,heterocycle, or is absent; R₅ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl,benzyl, phenyl, or heterocycle; or R₃ and R₅ combine to form aheterocyclic ring system; R₄ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, or heterocycle; and m is 0 or 1; or a pharmaceuticallyacceptable salt or solvate thereof.

In an embodiment of the third aspect of the invention, Q is CH₂, P is—O—, R₃ is H, Y—X is —CH₂NHCH₂— or —CH₂NHC(O)—, R₁ is selected from ahalogen, —OH or —OCH₃, R₂ is absent or selected from —OH and a halogen,R₄ is H or benzyl, and R₅ is H or benzyl, or a pharmaceuticallyacceptable salt or solvate thereof.

In another embodiment of the third aspect of the invention, Q is CH₂, Pis —O—, R₃ is CH₃, Y—X is —CH₂OCH₂—, R₁ is selected from a halogen, —OHor —OCH₃, R₂ is absent or selected from —OH and a halogen, R₄ is H orbenzyl, and R₅ is H or benzyl or a pharmaceutically acceptable salt orsolvate thereof.

In a particular embodiment of the third aspect of the invention, thecompound is5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In one embodiment,5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g.,atorvastatin).

In another embodiment of the third aspect of the invention, the compoundis5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In one embodiment,5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g.,atorvastatin).

In a further embodiment of the third aspect of the invention, thecompound is5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In one embodiment,5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g.,atorvastatin).

In an additional embodiment of the third aspect of the invention, thecell is a cardiovascular system cell, and in another embodiment of thethird aspect of the invention, the cell is in a tissue, and in a furtherembodiment, the cell or the tissue is in a subject.

In a fourth aspect, the invention features a method of modulating LDLRmRNA activity in a cell. This method includes contacting the cell withan effective amount of a compound having the formula:

where Y—X is —C(R₇)(R_(7a))N(R′)C(O)—; —C(R₇)(R_(7a))OC(O)—;—C(R₇)(R_(7a))N(R′)C(R₆)(R_(6a))—; —C(R₇)(R_(7a))S(O)₂C(R₆)(R_(6a))—;—S(O)₂C(R₆)(R_(6a))—; —C(R₇)(R_(7a))C(R₆)(R_(6a))—; —O—C(R₆)(R_(6a))—;—N(R′)C(R₆)(R_(6a))—; or —C(R₇)(R_(7a))OC(R₆)(R_(6a))—, where each ofR₆, R_(6a), R₇, and R_(7a), is, independently, selected from H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle, R′ is selectedfrom H, C₁₋₆ alkyl, benzyl, S(O)₂R″, and C(O)R″, and R″ is selected fromC₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle; when Q isH, P and R₃ are absent; when Q is CH₃, P and R₃ are absent; Q is CH₂, orC(O); when P is H, R₃ is absent; P is —O—, —N(R₈)(R_(8a)), or is absent,where the R₈ and R_(8a) are selected independently from the groupconsisting of H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, andheterocycle; R₁ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆ alkene,halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branched alkyl),—C(O)(R₉), —C(O)O(R₉), —C(O)N(R₉)(R_(9a)), or —S(O)₂N(R₉)(R_(9a)), whereR₉ and R_(9a) are selected independently H, C₁₋₆ alkyl, C₁₋₆fluoroalkyl, benzyl, phenyl, and heterocycle; R₂ is absent, C₁₋₆ alkyl,C₁₋₆ branched alkyl, C₁₋₆ alkene, halogen (F, Cl, Br, I), OH, —O—(C₁₋₆alkyl), —O—(C₁₋₆ branched alkyl), —C(O)(R₁₀), —C(O)O(R₁₀), or—C(O)N(R₁₀)(R_(10a)), where R₁₀ and R_(10a) are selected independentlyfrom H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle;or R₁ and R₂ are ortho substituents that together form a carbocyclic orheterocyclic ring system; R₃ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, heterocycle, or is absent; R₅ is H, C₁₋₆ alkyl, C₁₋₆fluoroalkyl, benzyl, phenyl, or heterocycle; or R₃ and R₅ combine toform a heterocyclic ring system; R₄ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl,benzyl, phenyl, or heterocycle; and m is 0 or 1; or a pharmaceuticallyacceptable salt or solvate thereof.

In some embodiments of the fourth aspect of the invention Q is CH₂, P is—O—, R₃ is H, Y—X is —CH₂NHCH₂— or —CH₂NHC(O)—, R₁ is selected from ahalogen, —OH or —OCH₃, R₂ is absent or selected from —OH and a halogen,R₄ is H or benzyl, and R₅ is H or benzyl, or a pharmaceuticallyacceptable salt or solvate thereof.

In other embodiments of the fourth aspect of the invention Q is CH₂, Pis —O—, R₃ is CH₃, Y—X is —CH₂OCH₂—, R₁ is selected from a halogen, —OHor —OCH₃, R₂ is absent or selected from —OH and a halogen, R₄ is H orbenzyl, and R₅ is H or benzyl or a pharmaceutically acceptable salt orsolvate thereof.

In a particular embodiment of the fourth aspect of the invention, thecompound is5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In one embodiment,5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g.,atorvastatin).

In another embodiment of the fourth aspect of the invention, thecompound is5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In one embodiment,5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g.,atorvastatin).

In a further embodiment of the fourth aspect of the invention, thecompound is5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In one embodiment,5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g.,atorvastatin).

In other embodiment of the fourth aspect of the invention, modulatingincludes inducing LDLR mRNA activity, and in another embodiment of thefourth aspect of the invention modulating includes stabilizing LDLR mRNAactivity.

In additional embodiments of the first aspect of the invention, themethod includes administering a second cholesterol lowering agent to thesubject. In some embodiments the second cholesterol lowering agent is alipase inhibitor, a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG CoA)reductase inhibitor, a HMG CoA synthase inhibitor, a cholesteryl estertransfer protein (CETP) inhibitor, a bile acid absorption inhibitor, acholesterol absorption inhibitor, a cholesterol synthesis inhibitor, asqualene synthetase inhibitor, a squalene epoxidase or cyclase inhibitoror a combination of both, a microsomal triglyceride transfer protein(MTP)/Apolipoprotein B (ApoB) secretion inhibitor, a fibrate, niacinalone or in combination with lovastatin, an ion-exchange resin, anantioxidant, an acyl coenzyme A:cholesterol acyltransferase (ACAT)inhibitor and a bile acid sequestrant, an HMG-CoA reductase geneexpression inhibitor, an HMG-CoA synthase gene expression inhibitor, ora proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitor. Inone embodiment,5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide is administered in combination with a statin (e.g., incombination with atorvastatin).

In further embodiments of the first aspect or the second aspect of theinvention, the subject, prior to administration of the compound ofFormula 1, was treated with a lipase inhibitor, a3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG CoA) reductase inhibitor, aHMG CoA synthase inhibitor, a cholesteryl ester transfer protein (CETP)inhibitor, a bile acid absorption inhibitor, a cholesterol absorptioninhibitor, a cholesterol synthesis inhibitor, a squalene synthetaseinhibitor, a squalene epoxidase or cyclase inhibitor or a combination ofboth, a microsomal triglyceride transfer protein (MTP)/Apolipoprotein B(ApoB) secretion inhibitor, a fibrate, niacin alone or in combinationwith lovastatin, an ion-exchange resin, an antioxidant, an acylcoenzymeA:cholesterol acyltransferase (ACAT) inhibitor and a bile acidsequestrant, an HMG-CoA reductase gene expression inhibitor, an HMG-CoAsynthase gene expression inhibitor, or a proprotein convertasesubtilisin kexin type 9 (PCSK9) inhibitor. In some embodiments, theprior administration did not effectively lower the cholesterol level inthe bloodstream of the subject.

In a fifth aspect, the invention features a use of a compound having theformula:

where Y—X is —C(R₇)(R_(7a))N(R′)C(O)—; —C(R₇)(R_(7a))OC(O)—;—C(R₇)(R_(7a))N(R′)C(R₆)(R_(6a))—; —C(R₇)(R_(7a))S(O)₂C(R₆)(R_(6a))—;—S(O)₂C(R₆)(R_(6a))—; —C(R₇)(R_(7a))C(R₆)(R_(6a))—; —O—C(R₆)(R_(6a))—;—N(R′)C(R₆)(R_(6a))—; or —C(R₇)(R_(7a))OC(R₆)(R_(6a))—, where each ofR₆, R_(6a), R₇, and R_(7a), is, independently, selected from H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle, R′ is selectedfrom H, C₁₋₆ alkyl, benzyl, S(O)₂R″, and C(O)R″, and R″ is selected fromC₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle; when Q isH, P and R₃ are absent; when Q is CH₃, P and R₃ are absent; Q is CH₂, orC(O); when P is H, R₃ is absent; P is —O—, —N(R₈)(R_(8a)), or is absent,where R₈ and R_(8a) are selected independently from H, C₁₋₆ alkyl, C₁₋₆fluoroalkyl, benzyl, phenyl, and heterocycle; R₁ is absent, C₁₋₆ alkyl,C₁₋₆ branched alkyl, C₁₋₆ alkene, halogen (F, Cl, Br, I), OH, —O—(C₁₋₆alkyl), —O—(C₁₋₆ branched alkyl), —C(O)(R₉), —C(O)O(R₉),—C(O)N(R₉)(R_(9a)), or —S(O)₂N(R₉)(R_(9a)), where R₉ and R_(9a) areselected independently from H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, and heterocycle; R₂ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl,C₁₋₆ alkene, halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆branched alkyl), —C(O)(R₁₀), —C(O)O(R₁₀), or —C(O)N(R₁₀)(R_(10a)), whereR₁₀ and R_(10a) are selected independently from H, C₁₋₆ alkyl, C₁₋₆fluoroalkyl, benzyl, phenyl, and heterocycle; or R₁ and R₂ are orthosubstituents that together form a carbocyclic or heterocyclic ringsystem; R₃ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl,heterocycle, or is absent; R₅ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl,benzyl, phenyl, or heterocycle; or R₃ and R₅ combine to form aheterocyclic ring system; R₄ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, or heterocycle; and m is 0 or 1; or a pharmaceuticallyacceptable salt or solvate thereof, in the production of a medicamentfor lowering the cholesterol level in the bloodstream of a subject, fortreating atherosclerosis, hypercholesterolemia, hypertriglyceridemia,diabetic complications, dyslipidemia, hyperlipidemia,hypoalphalipoproteinemia, metabolic syndrome, stroke, vascular dementia,chronic kidney disease, coronary heart disease, coronary artery disease,retinopathy, inflammation, thrombosis, peripheral vascular disease orcongestive heart failure, for inducing low density lipoprotein receptor(LDLR) expression in a cell or a tissue, or for inducing or stabilizingLDLR mRNA activity in a cell or a tissue.

In a particular embodiment of the fifth aspect of the invention, thecompound is5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.

In another particular embodiment of the fifth aspect of the invention,the compound is5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.

In a further particular embodiment of the fifth aspect of the invention,the compound is5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.

Definitions

The term “coronary artery disease,” as used herein, encompassesatherosclerotic plaque (e.g., prevention, regression, stabilization),vulnerable plaque (e.g., prevention, regression, stabilization),vulnerable plaque area (reduction), arterial calcification (e.g.,calcific aortic stenosis), increased coronary artery calcium score,dysfunctional vascular reactivity, vasodilation disorders, coronaryartery spasm, first myocardial infarction, myocardia re-infarction,ischemic cardiomyopathy, stent restenosis, PTCA restenosis, arterialrestenosis, coronary bypass graft restenosis, vascular bypassrestenosis, decreased exercise treadmill time, angina pectoris/chestpain, unstable angina pectoris, exertional dyspnea, decreased exercisecapacity, ischemia (reduce time to), silent ischemia (reduce time to),increased severity and frequency of ischemic symptoms, and reperfusionafter thrombolytic therapy for acute myocardial infarction.

The term “hypertension” encompasses lipid disorders with hypertension,systolic hypertension, and diastolic hypertension.

The term “peripheral vascular disease”, as used herein, encompassesperipheral vascular disease and claudication.

The term “diabetes,” as used herein, refers to any of a number ofdiabetogenic states including type I diabetes, type II diabetes,Syndrome X, Metabolic syndrome, lipid disorders associated with insulinresistance, impaired glucose tolerance, non-insulin dependent diabetes,microvascular diabetic complications, reduced nerve conduction velocity,reduced or loss of vision, diabetic retinopathy, increased risk ofamputation, decreased kidney function, kidney failure, insulinresistance syndrome, pluri-metabolic syndrome, central adiposity(visceral)(upper body), diabetic dyslipidemia, decreased insulinsensitization, diabetic retinopathy/neuropathy, diabeticnephropathy/micro and macro angiopathy and micro/macro albuminuria,diabetic cardiomyopathy, diabetic gastroparesis, obesity, increasedhemoglobin glycoslation (including HbAIC), improved glucose control,impaired renal function (dialysis or end stage), and impaired hepaticfunction (mild, moderate, or severe).

“Metabolic syndrome,” also known as “Syndrome X,” refers to a commonclinical disorder that is defined as the presence of increased insulinconcentrations in association with other disorders includinghyperlipidemia, dyslipidemia, viceral obesity, hyperglycemia,hypertension, and potentially hyperuricemis and renal dysfunction.

The term “Vitamin B₆” as used herein refers to one or more of threecompounds that are commonly referred to as vitamin B₆ namely pyridoxal,pyridoxamine, and pyridoxine. Pyridoxine differs from pyridoxamine bythe substituent at the ‘4’ position. Pyridoxine based on a pyridinering, with hydroxyl, methyl, and hydroxymethyl substituents and isconverted in vivo to pyridoxal 5-phosphate, the biologically active formof pyridoxine.

The terms “comprising” and “including” as used herein, are used in theiropen, non-limiting sense.

The term “C₁₋₆ alkyl,” as used herein, means saturated monovalenthydrocarbon radicals having straight or branched moieties and containingfrom 1 to 6 carbon atoms. The C₁₋₆ alkyl group may be substituted orunsubstituted. Examples of such groups include, but are not limited to,methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, and tertbutyl. Theterm “C₁₋₆ fluoroalkyl” refers to a C₁₋₆ alkyl substituted with one ormore fluorine atoms. Exemplary C₁₋₆ fluoroalkyl groups include, withoutlimitation, fluoromethyl, trifluoromethyl, and pentafluoroethyl. Theterm “C₁₋₆ branched alkyl” refers to alkyl group that include one ormore tertiary or quaternary carbon atoms.

By “C₁₋₆ alkenyl” is meant a branched or unbranched hydrocarbon groupcontaining one or more double bonds and having from 1 to 6 carbon atoms.For example, C₂ alkenyl may optionally include monocyclic or polycyclicrings, in which each ring desirably has from three to six members. TheC₂₋₄ alkenyl group may be substituted or unsubstituted.

By “carbocyclic group” or “carbocyclic ring” is meant a monocyclic orpolycyclic ring system which is saturated, partially unsaturated, orunsaturated (aromatic), and which consists of 3 to 8 carbon atoms(unless otherwise specified). Carbocyclic groups include alkyl groupssubstituted with such a monocyclic or polycyclic ring system. Exemplarycyclic groups include phenyl, benzyl, cyclopropyl, cyclobutyl,cyclopentyl, 2-phenylcyclopropane, and cyclohexyl. The carbocyclic groupmay be substituted or unsubstituted.

The terms “heterocycle” and “heterocyclic ring,” as used herein, meanaromatic or non-aromatic, monocyclic, bicyclic, tricyclic, tetracyclic,or spirocyclic group, having a total of from 3 to 10 atoms in its ringsystem, and containing from 2 to 9 carbon atoms and from one to fourheteroatoms each independently selected from O, S, and N and with theproviso that the ring of the group does not contain two adjacent O atomsor two adjacent S atoms. Furthermore, such heterocycle groups maycontain an oxo substituent at any available atom that will result in astable compound. For example, such a group may contain an oxo atom at anavailable carbon or nitrogen atom. Such a group may contain more thanone oxo substituent if chemically feasible. In addition, it is to beunderstood that when such a heterocycle group contains a sulfur atom,the sulfur atom may be oxidized with one or two oxygen atoms to affordeither a sulfoxide or sulfone. An example of a 4-membered heterocyclicgroup is azetidinyl (derived from azetidine). An example of a 5-memberedheterocyclic group is thiazolyl, and an example of a 10-memberedheterocyclic group is quinolinyl. Further examples of such heterocyclegroups include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl,homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl, andquinolizinyl. The heterocycle group may be substituted or unsubstituted.

As used herein, the terms “benzyl” and “phenyl” refer to bothsubstituted and unsubstitued benzyl and phenyl groups, respectively.

As used herein, the term “substituted” refers to a group (e.g., a “C₁₋₆alkyl,” “C₁₋₆ alkenyl,” “C₁₋₆ fluoroalkyl,” “benzyl,” “phenyl,”“heterocycle,” or “carbocyclic group”) in which one or more hydrogenatoms in the group are, independently, replaced with a substituentselected from, for example, methyl, ethyl, n-propyl, isopropyl, hydroxy,methoxy, ethoxy, fluorine, chlorine, bromine, iodine, cyano, nitro,amino, alkylamino, dialkylamino, carboxy, chloromethyl, trichloromethyl,trifluoromethyl, methoxyethyl, —CH₂C(O)NH₂, —C(O)CH₂N(CH₃)₂, —CH₂CH₂OH,—CH₂OC(O)NH₂, —CH₂CH₂NH₂, —CH₂CH₂CH₂NEt₂, —CH₂OCH₃, —C(O)NH₂, —C(NH)NH₂,—C(═NH)OEt, —C(O)NH-cyclopropyl, —C(O)NHCH₂CH₂OCH₃, —C(O)CH₂CH₂NHCH₃,—CH₂CH₂F, or —CH₂C(O)NHCH₃.

The term “solvate,” as used herein, means a pharmaceutically acceptablesolvate form of a compound of the present invention that retains thebiological effectiveness of such compound. Examples of solvates include,but are not limited to, compounds of the invention in combination withwater, isopropanol, ethanol, methanol, dimethylsulfoxide (DMSO), ethylacetate, acetic acid, ethanolamine, or mixtures thereof. It isspecifically contemplated that in the present invention one solventmolecule can be associated with one molecule of the compounds of thepresent invention, such as a hydrate. Furthermore, it is specificallycontemplated that in the present invention, more than one solventmolecule may be associated with one molecule of the compounds of thepresent invention, such as a dihydrate. Additionally, it is specificallycontemplated that in the present invention less than one solventmolecule may be associated with one molecule of the compounds of thepresent invention, such as a hemihydrate. Furthermore, solvates of thepresent invention are contemplated as solvates of compounds of thepresent invention that retain the biological effectiveness of thenon-hydrate form of the compounds.

A “pharmaceutically acceptable salt” as used herein means a salt thatretains the biological effectiveness of the free acids and bases of thespecified derivative, containing pharmacologically acceptable anions orcations, and is not biologically or otherwise undesirable. Examples ofpharmaceutically acceptable salts include, but are not limited to,acetate, acrylate, benzenesulfonate, benzoate (such as chlorobenzoate,methylbenzoate, dinitrobenzoate, hydroxybenzoate, and methoxybenzoate),bicarbonate, bisulfate, bisulfite, bitartrate, borate, bromide,butyne-1,4-dioate, calcium edetate, camsylate, carbonate, chloride,caproate, caprylate, clavulanate, citrate, decanoate, dihydrochloride,dihydrogenphosphate, edetate, edislyate, estolate, esylate,ethylsuccinate, formate, fumarate, gluceptate, gluconate, glutamate,glycollate, glycollylarsanilate, heptanoate, hexyne-1,6-dioate,hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,hydroxybutyrate, iodide, isobutyrate, isothionate, lactate,lactobionate, laurate, malate, maleate, malonate, mandelate, mesylate,metaphosphate, methane-sulfonate, methylsulfate, monohydrogenphosphate,mucate, napsylate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,nitrate, oleate, oxalate, pamoate (embonate), palmitate, pantothenate,phenylacetates, phenylbutyrate, phenylpropionate, phthalate,phospate/diphosphate, polygalacturonate, propanesulfonate, propionate,propiolate, pyrophosphate, pyrosulfate, salicylate, stearate,subacetate, suberate, succinate, sulfate, sulfonate, sulfite, tannate,tartrate, teoclate, tosylate, triethiodode, valerate salts, and cations,such as sodium, potassium, calcium, magnesium, ammonium, andtetraalkylammonium, among others.

The term “therapeutically effective amount,” as used herein, means anamount of a compound of the present invention, or a pharmaceuticallyacceptable salt or solvate thereof, that, when administered to a mammalin need of such treatment, is sufficient to effect treatment, as definedherein.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired result, e.g., increaseof LDLR levels in a cell or increase expression of a functional LDLRreceptor in a cell. The increase of LDLR levels or increase inexpression of a functional LDLR receptor may be one and a half-fold,two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold,eight-fold, nine-fold, ten-fold, eleven-fold, twelve-fold, orfifteen-fold relative to the level prior to contacting the cell with acompound of the present invention or a pharmaceutically acceptable saltor solvate thereof.

The term “LDL-lowering amount,” as used herein, refers to an amount of acompound of the present invention, or a pharmaceutically acceptable saltor solvate thereof, that, when administered to a mammal in need of suchtreatment, lowers the serum LDL level as compared to the serum LDL levelprior to treatment. The serum LDL level may be one and a half-fold,two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold,eight-fold, nine-fold, ten-fold, eleven-fold, twelve-fold, orfifteen-fold lower relative to the serum LDL level prior to treatment.The serum LDL level may be measured using standard methods, e.g., alipid panel.

The term “tissue,” as used herein, refers to a group or layer ofsimilarly specialized cells that together perform certain specialfunctions. Examples of tissues include liver, blood, adipose tissue,fatty tissue, subcutaneous tissue, and muscular tissue.

The term “organ,” as used herein, refers to a group of tissues in aliving organism, e.g., mammal, that have been adapted to perform aspecific function. In mammals, for example, organs are grouped intoorgan systems, e.g., the esophagus, stomach, and liver are organs of thedigestive system.

The term “cardiovascular system cell,” as used herein, refers to a cellthat is a part of the cardiovascular system that conveys blood throughvessels to and from all parts of the body, carrying nutrients and oxygento tissues and removing carbon dioxide and other wastes. Acardiovascular system cell may be part of a blood vessel or may be partof the blood itself.

The term “heterozygous familial hypercholesterolemia,” as used herein,refers to a genetic condition that causes high low-density lipoprotein(LDL) cholesterol, where the subject has one familialhypercholesterolemia gene mutated on one allele.

The term “homozygous familial hypercholesterolemia,” as used herein,refers to a genetic condition that causes high low-density lipoprotein(LDL) cholesterol, where the subject has one familialhypercholesterolemia gene mutated on both alleles.

Polygenic familial hypercholesterolemia and polygenichypercholesterolemia refer to a genetic condition that causes highlow-density lipoprotein (LDL) cholesterol, where the subject has severalgenes contributing to the disease.

The term “pharmaceutically acceptable formulation,” as used herein,means a combination of a compound of the invention, or apharmaceutically acceptable salt or solvate thereof, and a carrier,diluent, and/or excipients that are compatible with a compound of thepresent invention, and is not deleterious to the recipient thereof.Pharmaceutical formulations can be prepared by procedures known to thoseof ordinary skill in the art. For example, the compounds of the presentinvention can be formulated with common excipients, diluents, orcarriers, and formed into tablets, capsules, and the like. Examples ofexcipients, diluents, and carriers that are suitable for suchformulations include the following: fillers and extenders such asstarch, sugars, mannitol, and silicic derivatives; binding agents suchas carboxymethyl cellulose and other cellulose derivatives, alginates,gelatin, and polyvinyl pyrrolidone; moisturizing agents such asglycerol; disintegrating agents such as povidone, sodium starchglycolate, sodium carboxymethylcellulose, agar, calcium carbonate, andsodium bicarbonate; agents for retarding dissollution such as paraffin;resorption accelerators such as quaternary ammonium compounds; surfaceactive agents such as cetyl alcohol, glycerol monostearate; adsorptivecarriers such as keolin and bentonite; and lubricants such as talc,calcium and magnesium stearate and solid polyethylene glycols. Finalpharmaceutical forms may be pills, tablets, powders, lozenges, saches,cachets, or sterile packaged powders, and the like, depending on thetype of excipient used. Additionally, it is specifically contemplatedthat pharmaceutically acceptable formulations of the present inventioncan contain more than one active ingredient. For example, suchformulations may contain more than one compound according to the presentinvention. Alternatively, such formulations may contain one or morecompounds of the present invention and one or more additionalcholesterol modulating agents. A pharmaceutically acceptable formulationmay also include but is not limited to compounds, other than thecompounds of formula I, having a structure such that, uponadministration to a recipient or patient, a compound of this invention,active metabolite, or residue thereof is directly or indirectlyprovided.

The terms “treat,” “treating,” and “treatment” include: (i) preventing adisease or condition from occurring in a subject which may bepredisposed to the condition, such that the treatment constitutesprophylactic treatment for the pathologic condition; (ii) modulating orinhibiting a disease or condition, i.e., arresting its development;(iii) relieving a disease or condition, i.e., causing regression of thedisease or condition; or (iv) relieving and/or alleviating a disease orcondition or the symptoms resulting from the disease or condition, e.g.,relieving an inflammatory response without addressing the underlyingdisease or condition.

The terms “co-administration,” “co-administering,” “co-administer,”“co-administered,” or “combination therapy,” as used herein, refer tothe administration of a combination of at least a first agent and asecond agent or two or more agents according to the present invention.Such co-administration can be performed such that two or multiple agentsare part of the same composition or part of the same unitary dosageform. Co-administration also; includes administering a first agent and asecond agent, or more than two agents separately and as part of the sametherapeutic regimen. The agents, if administered separately, need notnecessarily be administered at essentially the same time, although theycan be if so desired. Thus co-administration includes, for example,administering a first agent and a second agent as separate dosages ordosage forms, but at the same time. Co-administration also includesseparate administration at different times and in any order.

The term “compound of the present invention,” refers to any of theabove-mentioned compounds, as well as those in the Examples that follow,and include those generically described or those described as species.The term also refers to pharmaceutically acceptable salts or solvates ofthese compounds.

The abbreviations used herein refer to the following:

Abbreviation Definition AcOH Acetic acid Ar Argon BSA Bovine serumalbumin DMF Dimethylformamide DNA Deoxyribonucleic acid EtOH Ethylalcohol, ethanol g Gram HPLC High pressure liquid chromatography M MolarMeOH Methyl alcohol, methanol mg Milligram mp Melting point min MinutemL Milliliter mmol Millimole nM Nanomolar RNA Ribonucleic acid THFTetrahydrofuran LDLR Low density lipoprotein receptor LDL Low densitylipoprotein μM micromolar nM nanomolar

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a synthetic scheme showing the synthesis of compound VI.

FIG. 2 is a synthetic scheme showing the synthesis of compound IX.

FIG. 3 is a synthetic scheme showing the synthesis of compound XI.

FIG. 4 is a synthetic scheme showing the synthesis of compound XIX.

FIG. 5 is a synthetic scheme showing the synthesis of compounds XXII andXXIII.

FIG. 6 is a synthetic scheme showing the synthesis of compound XXXII.

FIG. 7 is a synthetic scheme showing the synthesis of compound XXXV.

FIG. 8 is a synthetic scheme showing the synthesis of compound XXXIX.

FIG. 9 is a synthetic scheme showing the synthesis of compound XLV.

FIG. 10 is a synthetic scheme showing the synthesis of compound XLVII.

FIG. 11 is a synthetic scheme showing the synthesis of compound L.

FIG. 12 is a synthetic scheme showing the synthesis of compound LVI.

FIG. 13A to FIG. 13C are a series of images and graphs showing: (A andB) Western blot analysis and relative quantitation of LDLR protein ofCompounds 1, 2, 3, and 21, in the HepG2 cell line; (C) dose responseeffect of Compound 2 in HepG2 cells.

FIG. 14 is a series of images showing the immunofluorescence analysis ofLDLR protein using confocal microscopy.

FIG. 15 is a series of graphs showing the effects of Compound 2 on LDLRprotein levels by Western blot analyses in various cell types.

FIG. 16 is a graph showing the relative quantities of LDLR, HMGCR, andPCSK9 mRNA in cells treated with Compounds 1, 2, 3, and 21.

FIG. 17 is a series of graphs showing the effects of Compound 2 on LDLRpromoter or LDLR 3′UTR of the Human LDLR gene.

FIG. 18 is a series of graphs showing the effects of Compound 2 on mRNAdecay or the Human LDLR and Human GAPDH genes.

FIG. 19 is a series of graphs showing the effects of Compound 2 anddifferent combined inhibitors on LDLR protein levels by Western blotanalyses in HepG2 cells.

FIG. 20 is a series of graphs showing Pharmacokinetics of Compound 2 inadult Sprague-Dawley rats.

DETAILED DESCRIPTION

The present disclosure describes methods for lowering LDL-cholesterollevel in a subject. The methods can include administering to the subjecta LDL-lowering amount of a compound of Formula I. Further, methods areprovided for inducing low density lipoprotein receptor (LDLR) expressionin a cell, e.g., a cell in a tissue or an organ), and for modulatingLDLR mRNA or protein expression and LDLR activity in a cell byadministering compound of Formula I.

Screening of available compound libraries yielded potent inducers oflow-density lipoprotein receptor (LDLR) in hepatic cells, selected fromcompounds described in U.S. Pat. No. 8,742,123 B2.

Compounds

One aspect of the invention provides a compound having the Formula I, ora pharmaceutically acceptable salt thereof:

In some embodiments, Y—X is —C(R₇)(R_(7a))N(R′)C(O)—;—C(R₇)(R_(7a))OC(O)—; —C(R₇)(R_(7a))N(R′)C(R₆)(R_(6a))—;—C(R₇)(R_(7a))S(O)₂C(R₆)(R_(6a))—; —S(O)₂C(R₆)(R_(6a))—;—C(R₇)(R_(7a))C(R₆)(R_(6a))—; —O—C(R₆)(R_(6a))—; —N(R′)C(R₆)(R_(6a))—;or —C(R₇)(R_(7a))OC(R₆)(R_(6a))—. Each of R₆, R_(6a), R₇, and R_(7a),can be, independently, selected from H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl,benzyl, phenyl and heterocycle, R′ can be selected from H, C₁₋₆ alkyl,benzyl, S(O)₂R″, and C(O)R″, and R″ may be selected from C₁₋₆ alkyl,C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle;

In some embodiments, when Q is H, P and R₃ are absent. In otherembodiments, when Q is CH₃, P and R₃ are absent. In some embodiments, Qis CH₂, or C(O).

In some embodiments, when P is H, R₃ is absent. In other embodiments, Pis —O—, —N(R₈)(R_(8a)), or is absent, where the R₈ and R_(8a) areselected independently from the group consisting of H, C₁₋₆ alkyl, C₁₋₆fluoroalkyl, benzyl, phenyl, and heterocycle.

In some embodiments, R₁ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆alkene, halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branchedalkyl), —C(O)(R₉), —C(O)O(R₉), —C(O)N(R₉)(R_(9a)), or—S(O)₂N(R₉)(R_(9a)). R₉ and R_(9a) can be selected independently fromthe group consisting of H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyland heterocycle.

In some embodiments, R₂ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆alkene, halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branchedalkyl), —C(O)(R₁₀), —C(O)O(R₁₀), or —C(O)N(R₁₀)(R_(10a)). R₁₀ andR_(10a) can be selected independently from the group consisting of H,C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl, and heterocycle; or R₁ andR₂ can be ortho substituents that together form a carbocyclic orheterocyclic ring system.

In some embodiments, R₃ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, heterocycle, or is absent.

In some embodiments, R₅ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, or heterocycle; or R₃ and R₅ combine to form a heterocyclic ringsystem.

In some embodiments, R₄ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, or heterocycle.

In some embodiments, m is 0 or 1.

In some embodiments, Q is CH₂, P is —O—, R₃ is H, Y—X is —CH₂NHCH₂— or—CH₂NHC(O)—. In some embodiments, R₁ is selected from a halogen, —OH or—OCH₃, R₂ is absent or selected from —OH and a halogen. In someembodiments, R₄ is H or benzyl, and R₅ is H or benzyl, or apharmaceutically acceptable salt or solvate thereof.

In some embodiments, Q is CH₂, P is —O—, R₃ is CH₃, Y—X is —CH₂OCH₂—. Insome embodiments, R₁ is selected from a halogen, —OH or —OCH₃, R₂ isabsent or selected from —OH and a halogen. In some embodiments, R₄ is Hor benzyl, and R₅ is H or benzyl or a pharmaceutically acceptable saltor solvate thereof.

In some embodiments, the compound of Formula I is5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In some embodiments, the compound of Formula I is5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide. In some embodiments, the compound of Formula I is5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.

Pharmaceutical Compositions

Pharmaceutical compositions contemplated herein include at least onecompound of the present invention, and pharmaceutically acceptablesalts, solvate or formulation thereof, with a pharmaceuticallyacceptable carrier, adjuvant, or vehicle. Pharmaceutically acceptablecarriers, adjuvants and vehicles that may be used in the pharmaceuticalcompositions of this invention include, but are not limited to, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethyleneglycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol,liposomes, and wool fat.

Compounds of the present invention that are basic may be prepared as asalt using suitable methods known in the art, including treatment of thefree base with an inorganic acid, such as hydrochloric acid; hydrobromicacid; sulfuric acid; nitric acid; phosphoric acid; and the like, or withan organic acid, such as acetic acid; maleic acid; succinic acid;mandelic acid; fumaric acid; malonic acid; pyruvic acid; oxalic acid;glycolic acid; salicylic acid; pyranosidyl acid, such as glucuronic acidor galacturonic acid; alpha-hydroxy acid, such as citric acid ortartaric acid; amino acid, such as aspartic acid or glutamic acid;aromatic acid, such as benzoic acid or cinnamic acid; sulfonic acid,such as p-toluenesulfonic acid or methanesulfonic acid, and the like.

It is understood by those skilled in the art that the compounds of thepresent invention, salts, or solvates thereof may exist in differentcrystal or polymorphic forms that are within the scope of the presentinvention and specified formulas.

Basic compounds of the present invention can form a variety of saltswith various inorganic and organic acids. Although such salts must bepharmaceutically acceptable for administration to animals, it is commonpractice to first isolate the compound of the present invention as apharmaceutically unacceptable salt and then convert to a free basecompound by treatment with an alkaline reagent and subsequently convertthe latter free base to a pharmaceutically acceptable acid additionsalt. The acid addition salts of the base compounds of this inventioncan be prepared by treating the base compound with a substantiallyequivalent amount of the selected mineral or organic acid in an aqueoussolvent medium or in a suitable organic solvent, such as methanol orethanol.

Compounds of the present invention that are acidic may be prepared as asalt using suitable methods known in the art, including treatment of thefree acid with an inorganic or organic base, such as an amine (primary,secondary, or tertiary); an alkali metal or alkaline earth metalhydroxide; or the like. Examples of suitable salts include organic saltsderived from amino acids such as glycine and arginine; ammonia; primary,secondary, and tertiary amines; and cyclic amines, such as piperidine,morpholine, and piperazine; as well as inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum, and lithium.

Acidic compounds of the present invention can form base salts withvarious pharmacologically acceptable cations. Examples of such saltsinclude the alkali metal or alkaline-earth metal salts and particularly,the sodium and potassium salts, which can be prepared using conventionaltechniques. The chemical bases suitable as reagents in preparing thepharmaceutically acceptable base salts of this invention are those whichform non-toxic base salts with the acidic compounds of the presentinvention. Such non-toxic base salts include those derived from suchpharmacologically acceptable cations as sodium, potassium, calcium, andmagnesium, etc. These salts can be prepared by treating thecorresponding acidic compounds with an aqueous solution containing thedesired pharmacologically acceptable cations, and then evaporating theresulting solution to dryness, preferably under reduced pressure.Alternatively, they may also be prepared by mixing lower alkanolicsolutions of the acidic compounds and the desired alkali metal alkoxidetogether, and then evaporating the resulting solution to dryness in thesame manner as before. In either case, stoichiometric quantities ofreagents are preferably employed in order to ensure completeness ofreaction and maximum yield of the desired final product.

To treat or prevent diseases or conditions caused or mediated byelevated cholesterol, a pharmaceutical composition, including at leastone of the compounds of the present invention, is administered in apharmaceutically acceptable formulation prepared by combining atherapeutically effective amount of the compound with one or morepharmaceutically suitable carriers including diluents, excipients andauxiliaries that facilitate processing of the active compounds into apharmaceutically acceptable formulation. Carriers employed may be eithersolid or liquid. Exemplary solid carriers are lactose, sucrose, talc,gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and thelike. Exemplary liquid carriers are syrup, peanut oil, olive oil, water,and the like. Similarly, the inventive compositions may includetime-delay or time-release material known in the art, such as glycerylmonostearate or glyceryl distearate alone or with a wax, ethylcellulose,hydroxypropylmethylcellulose, methylmethacrylate or the like. Furtheradditives or excipients may be added to achieve the desired formulationproperties. For example, a bioavailability enhancer, such: as Labrasol®(a nonionic oil-in-water surfactant), Gelucire® (a nonionicwater-dispersible surfactant), or the like, or formulator, such as CHIC(carboxy-methylcellulose), PG (propyleneglycol), or PEG(polyethyleneglycol), may be added. Gelucire®, a semi-solid vehicle thatprotects active ingredients from light, moisture and oxidation, may beadded, e.g., when preparing a capsule formulation.

If a solid carrier is used, the preparation can be tableted, placed in ahard gelatin capsule in powder or pellet form, or formed into a trocheor lozenge. The amount of solid carrier may vary, but generally will befrom about 25 mg to about 1 g. If a liquid carrier is used, thepreparation may be in the form of syrup, emulsion, soft gelatin capsule,sterile injectable solution or suspension in an ampoule or vial ornon-aqueous liquid suspension. The inventive compositions are preparedin unit-dosage form appropriate for the mode of administration, e.g.,parenteral or oral administration.

To obtain a stable water-soluble dose form, a pharmaceuticallyacceptable salt of a compound of the present invention may be dissolvedin an aqueous solution of an organic or inorganic acid, such as 0.3 Msolution of succinic acid or citric acid. If a soluble salt form is notavailable, the agent may be dissolved in a suitable co-solvent orcombinations of co-solvents. Examples of suitable co-solvents includealcohol, propylene glycol, polyethylene glycol 300, polysorbate 80,glycerin, and the like, in concentrations ranging from 0-60% of thetotal volume. In an exemplary embodiment, a compound of the presentinvention is dissolved in DMSO and diluted with water. The compositionmay also be in the form of a solution of a salt form of the activeingredient in an appropriate aqueous vehicle such as water or isotonicsaline or dextrose solution.

Pharmaceutical preparations for oral use can be obtained using a solidexcipient in an admixture with the active ingredient (agent), optionallygrinding the resulting mixture, and processing the mixture of granulesafter adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients include: fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; and cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

The pharmaceutical compositions, including the compounds of the presentinvention may also contain suitable solid- or gel-phase carriers, orexcipients. These carriers and excipients may provide marked improvementin the bioavailability of poorly soluble drugs. Examples of suchcarriers or excipients include calcium carbonate, calcium, phosphate,sugars, starches, cellulose derivatives, gelatin, and polymers such aspolyethylene glycols. Furthermore, additives or excipients such asGelucire® (a nonionic water-dispersible surfactant), Capryol® (anonionic water-insoluble surfactant), Labrafil® (a nonionicwater-dispersible surfactant), Labrasol® (a nonionic oil-in-watersurfactant), Lauroglycol® (a nonionic water-insoluble surfactant),Plurol® (a nonionic water-in-oil emulsifier), Peceol® (an oily liquidvehicle), Transcutol® (a high-purity solvent), and the like, may beused. Further, the pharmaceutical composition may be incorporated into askin patch for delivery of the drug directly onto the skin.

The compounds of this invention may be modified by appending appropriatefunctionalities to enhance selective biological properties. Suchmodifications are known in the art and include those which increasebiological penetration into a given biological system (e.g., blood,lymphatic system, central nervous system), increase oralbioavailability, increase solubility to allow administration byinjection, alter metabolism or alter rate of excretion (PharmacokineticOptimization in Drug Research, Testa, B. et al, 2001, Wiley-VCH, VCHA).

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally, or via an implanted reservoir and are preferablyadministered orally or parenterally. The pharmaceutical compositions ofthis invention may contain any conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants or vehicles. The term “parenteral” or“parenterally” as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intrasynovial, intrasternal,intrathecal, intralesional, and intracranial injection or infusiontechniques.

Pharmaceutical compositions of the invention may be in the form of asterile injectable preparation, for example, as a sterile injectableaqueous or oleaginous suspension. This suspension may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents (for example, Tween 80) and suspending agents. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solutions. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions.

Pharmaceutical compositions of the invention may be orally administeredin any orally acceptable dosage form including, but not limited to,capsules, tablets, and aqueous suspension and solutions. In the case oftablets for oral and carriers which are commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried corn starch. When aqueous suspensionsare administered orally, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening and/orflavoring and/or coloring agents may be added.

Pharmaceutical compositions of the invention may also be administered inthe form of suppositories for rectal administration. These compositionscan be prepared by mixing a compound of this invention with a suitablenon-irritating excipient which is solid at room temperature but liquidat the rectal temperature and therefore will melt in the rectum torelease the active components. Such materials include, but are notlimited to, cocoa butter, beeswax, and polyethylene glycols.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well known in the art of pharmaceutical formulation and maybe prepared as solutions in saline employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

Dosages

It will be appreciated that the actual dosages of the agents of thisinvention will vary according to the particular agent being used, theparticular composition formulated, the mode of administration, and theparticular site, host, and disease being treated. Those skilled in theart using: conventional dosage determination tests in view of theexperimental data for a given compound may ascertain optimal dosages fora given set of conditions. For oral administration, an exemplary dailydose generally employed will be from about 0.001 to about 1000 mg/kg ofbody weight, with courses of treatment repeated at appropriateintervals, preferably between 0.01 and about 25 mg/kg body weight perday, and more preferably between about 0.5 and about 25 mg/kg bodyweight per day of the active ingredient compound are useful in theprevention and treatment of elevated cholesterol, including highcirculating LDL.

Furthermore, the pharmaceutically acceptable formulations of the presentinvention may contain a compound of the present invention, or apharmaceutically acceptable salt or solvate thereof, in an amount ofabout 10 mg to about 2000 mg, or from about 10 mg to about 1500 mg, orfrom about 10 mg to about 1000 mg, or from about 10 mg to about 750 mg,or from about 10 mg to about 500 mg, or from about 25 mg to about 500mg, or from about 50 to about 500 mg, or from about 100 mg to about 500mg. Additionally, the pharmaceutically acceptable formulations of thepresent invention may contain a compound of the present invention, or apharmaceutically acceptable salt or solvate thereof, in an amount fromabout 0.5 w/w % to about 95 w/w %, or from about 1 w/w % to about 95 w/w%, or from about 1 w/w % to about 75 w/w %, or from about 5 w/w % toabout 75 w/w %, or from about 10 w/w % to about 75 w/w %, or from about10 w/w % to about 50 w/w %.

Typically, the pharmaceutical compositions of this invention will beadministered from about 1 to about 5 times per day or alternatively, asa continuous infusion. Such administration can be used as a chronic oracute therapy. The amount of active ingredient that may be combined withthe carrier materials to produce a single dosage form will varydepending upon the patient treated and the particular mode ofadministration. A typical preparation will contain from about 5% toabout 75% active compound (w/w). Preferably, such preparations containfrom about 20% to about 50% active compound.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. When thesymptoms have been alleviated to the desired level, treatment shouldcease, at least in principle. Patients may, however, requireintermittent treatment on a long-term basis, upon any recurrence ofdisease symptoms, especially for high elevations of cholesterol.

As the skilled artisan will appreciate, precision medicine, lower orhigher doses than those recited above may be required. Specific dosageand treatment regimen for any particular patient will depend upon avariety of factors, including the genetic background, activity of thespecific compound employed, the age, body weight, general health status,sex, diet, time of administration, rate of excretion, drug combination,the severity and course of the infection, the patient's disposition tothe infection and the judgment of the treating physician.

With respect to the compounds of the present invention, the particularpharmaceutical formulation, the dosage, and the number of doses givenper day to a mammal requiring such treatment, are all choices within theknowledge of one of ordinary skill in the art and can be determinedwithout undue experimentation.

Treatment

The compounds of this invention are also useful as commercial reagentswhich effectively lower circulating cholesterol. As commercial reagent,the compounds of this invention, and their derivatives, may bederivatized to bind to a stable resin as a tethered substrate foraffinity chromatography applications. These and other uses whichcharacterize commercial cholesterol reducing agents will be evident tothose of ordinary skill in the art.

The compounds of the present invention can be used alone (i.e.,monotherapy) or administered in combination with one or more othercholesterol reducing agents, in the treatment of, for example,atherosclerosis (lipid-associated cardiovascular disorders, e.g., U.S.Pat. No. 4,891,220), hypercholesterolemia (lipid-associatedcardiovascular disorder, e.g., U.S. Pat. No. 4,891,220),hypertriglyceridemia (a high level of triglycerides in the blood, e.g.,U.S. Pat. No. 8,293,727), diabetic complications (include, for example,diabetic retinopathy, nephropathy, and neuropathy, resulting fromabnormalities in microvascular function, e.g., U.S. Pat. No. 5,281,619),dyslipidemia (an abnormally elevated cholesterol or lipids in the blood,e.g., U.S. Pat. No. 6,630,450), hyperlipidemia (lipid-associatedcardiovascular disorder, e.g., U.S. Pat. No. 4,891,220),hypoalphalipoproteinemia (a high-density lipoprotein deficiency, e.g.,U.S. Pat. No. 6,147,089), metabolic syndrome (obesity associated withhypertension, glucose intolerance, atherosclerosis, and dyslipidemia,e.g., U.S. 2006/0211020), stroke (damage to the brain from interruptionof its blood supply, e.g., U.S. Pat. No. 5,385,940), vascular dementia(brain damage caused by multiple strokes, e.g., U.S. Pat. No.6,458,807), chronic kidney disease (longstanding disease of the kidneysleading to renal failure, e.g., U.S. 2009/0081713), coronary heartdisease (damage or disease in the heart's major blood vessels, e.g.,U.S. Pat. No. 6,242,186), coronary artery disease (damage or disease inthe heart's major blood vessels, e.g., U.S. Pat. No. 5,036,857),retinopathy (a complication of diabetes that affects the eyes, e.g.,U.S. Pat. No. 5,019,591), inflammation (a response triggered by damageto living tissues, e.g., U.S. Pat. No. 6,136,839), thrombosis (formationof a blood clot inside a blood vessel, obstructing the flow of bloodthrough the circulatory system, e.g., U.S. Pat. No. 4,880,788),peripheral vascular disease (a circulatory condition in which narrowedblood vessels reduce blood flow to the limbs, e.g., U.S. Pat. No.8,143,316), or congestive heart failure (heart's inability to pump bloodsufficiently to maintain blood flow to meet the body's needs, e.g., U.S.Pat. No. 5,935,924) in a mammal.

The compounds of this invention may be administered in combination withcholesterol reducing agents which target other steps in the cholesterolmetabolism. These agents are, for example, a lipase inhibitor (asubstance used to reduce the activity of lipases found in the intestine,e.g., orlistat; e.g., U.S. Pat. No. 6,558,690), a3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG CoA) reductase inhibitor (astatin that facilitates the prevention of cardiovascular disease bylowering low-density lipoprotein cholesterol, e.g., atorvastatin,fluvastatin, lovastatin; e.g., Hong et al. Healthc. Inform. Res. 2017,3, 199), a HMG CoA synthase inhibitor (inhibits the HMG-CoA synthaseenzyme that is an intermediate in cholesterol synthesis and ketogenesis;e.g., Skaff et al. Biochemistry 2012, 51, 4713), a cholesteryl estertransfer protein (CETP) inhibitor (inhibits the promotion of thetransfer of cholesteryl esters from HDL to very low-density lipoproteinand LDL, e.g., anacetrapib, evacetrapib, dalcetrapib; e.g., Kosmas etal. Clin. Med. Insights Cardiol. 2016; 10, 37), a bile acid absorptioninhibitor (reduces low density lipoprotein (LDL) cholesterol levels,e.g., cholestyramine, colestipol, colesevelam; e.g., Kramer et at. Curr.Med. Chem. 2006, 13, 997), a cholesterol absorption inhibitor (preventsthe uptake of cholesterol from the small intestine into the circulatorysystem, e.g., ezetimibe; e.g., Dujovne et al. Am. J. Cardiol. 2002, 90,1092), a cholesterol synthesis inhibitor (HMG-CoA reductase inhibitors,e.g., statins; e.g., Endo Atheroscler. Suppl. 2004, 3, 67), a squalenesynthase inhibitor (decreases cholesterol synthesis and plasmatriglyceride levels, e.g., zaragozic acids,2,8-dioxabicyclo[3.2.1]octane derivatives; e.g., Kourounakis et al.Curr. Med. Chem. 2011, 18, 4418), a squalene epoxidase (enzyme thatoxidizes squalene to squalene epoxide; e.g., Ryder et al. Biochem. J.1985, 230, 765) or cyclase inhibitor (an enzyme that has a diverse rolein cell regulation and activity; e.g., Wang et al. Sci. Transl. Med.2011, 3, 65ra3) or a microsomal triglyceride transfer protein (MTP)inhibitor or Apolipoprotein B (ApoB) secretion inhibitor, or acombination of both (inhibits lipoprotein assembly; e.g., Hussain et al.J. Lipid Res. 2003, 44, 22), a fibrate (a hypolipidemic agent, e.g.,choline fenofibrate; e.g., Shepherd Postgrad. Med. J. 1993, 69, S34),niacin (lipid-lowering medication, e.g., Canner et al. J. Am. Col.Cardiol. 1986, 8, 1245) alone or in combination with lovastatin (astatin that treats high cholesterol and triglyceride levels; e.g., Downset al. JAMA 1998, 279, 1615), an ion-exchange resin (e.g.,colestyramine, is a bile acid sequestrant; e.g., Hashim et al. JAMA1965, 192, 289), an antioxidant (used to reducing the levels of LDLcholesterol; e.g., Anderson et al. N. Engl J. Med. 1995, 332, 488), anacyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor (inhibitsan intracellular enzyme that catalyzes the formation of cholesterolesters from cholesterol and fatty acyl-coenzyme; e.g., Chang et al. Am.J. Physiol. Endocrinol. Metab. 2009, 297, E1) and a bile acidsequestrant (polymeric resin that serve as ion-exchange resins used tobind components of bile in the gastrointestinal tract, e.g., colestipol;e.g., Ast et al. Clin. Pharmacol. 1990, 30, 99), an HMG-CoA reductasegene expression inhibitor, an HMG-CoA synthase gene expression inhibitor(SREBP2 inhibitors would strongly decrease gene expression of HMG-CoAreductase and synthase; Pandyra et al. Oncotarget. 2015, 6(29):26909-26921; Pandyra et al. Cancer Res. 2014, 74(17): 4772-82; statinsare HMG-CoA reductase inhibitors and, therefore, increase HMG-CoAreductase and synthase gene expression by a feedback mechanism (mediatedby the transcription factor SREBP-2) but their major actions are viaupregulation of LDLR mRNA (also by SREBP-2); a list of the suitabledrugs for combination are listed in Table 1) or a proprotein convertasesubtilisin kexin type 9 (PCSK9) inhibitor (decreases LDL blood levels,e.g., alirocumab, evolocumab; e.g., N. Engl. J. Med. 2015, 373, 1588) orcombination with other lipid-lowering drugs such as inhibitors ofAPOCIII, ANGPTL3, ANGPTL4, which increase lipoprotein lipase activity,gemecabene (Gemphire) or bempedoic acid (Esperio).

Combination therapies according to this invention exert an additive orcombined effect on elevated cholesterol reduction because eachtherapeutic agent of the combination acts on a different site ofcholesterol metabolism. The use of such combination therapies alsoadvantageously enables a reduction in the dosage of each elevatedcholesterol reducing agent, compared to administration of either agentalone as a monotherapy, while providing an equivalent or bettertherapeutic or prophylactic effect. Administration of lower doses ofeach therapeutic agent often reduces or even eliminates side effects ortoxicity relative to monotherapy. Further, combination therapies reducethe potential for the development of undesired side effects to theagents administered compared to monotherapy.

Administration of the compounds of this invention in combinationtherapies with other agents to patients may be sequential or concurrent.Further, pharmaceutical or prophylactic compositions of this inventionmay include a combination of cholesterol reducing agent of thisinvention and another therapeutic or prophylactic agent. Additionalexamples of agents useful for treating elevated cholesterol and suitablefor combination therapies with the compounds of this invention arelisted in Table 1.

TABLE 1 Drug Type Generic Name (brand name) Indication De novocholesterol Atorvastatin (Lipitor ®), For Management as an adjunct todiet synthesis inhibitors Fluvastatin (Lescol ®, Lescol to reduceelevated total-C, LDL-C, (statins or HMG-CoA XL), Lovastatin (Mevacor ®,apo B and TG levels in patients with reductase inhibitors) Altropev),Pravastatin primary hypercholesterolemia and mixed (Pravachol ®),Rosuvastatin dyslipidemia. For primary prevention (Crestor ®),Simvastatin of coronary heart disease and to slow (Zocor ®),Pitavastatin progression of coronary atherosclerosis (Livalo ®) inpatients with coronary heart disease. Inhibitors of Ezetimibe (Zetia ®),For use as adjunctive therapy to diet intestinal cholesterol Ezetimibe +simvastatin for the reduction of elevated total-C, absorption(Vytorin ®) LDL-C, and Apo B in patients with (NPC1L1 inhibitors)primary (heterozygous familial and non-familial) hypercholesterolemiaPPAR agonists Gemfibrozil (Lopid ®), For the treatment of primary(Fibrates) Fenifibrate (Antara ®, hyperlipidaemia types IIa, IIb, III,Lofibra ®, Tricor ®), IV, and V (Fredrickson classification) Clofibrate(Atromid ®) corresponding to groups I, II, and III of the EuropeanAtherosclerosis Society guidelines - when diet alone or improvements inlifestyle such as increases exercise or weight reduction do not lead toan adequate response. Also for the treatment of secondaryhyperlipidaemias, e.g. severe hypertriglyceridemias, when sufficientimprovement does not occur after correction of the underlying disorder(e.g. diabetes mellitus) Bile Cholestyramine (Questran ®, Indicated asadjunctive therapy to diet sequestrants (resins) Prevalite ®),Colestipol for the reduction of elevated serum (Colestid ®, Flavoredcholesterol in patients with primary Colestid), Colesevelamhypercholesterolemia (elevated low (Welchol ®) density lipoprotein [LDL]cholesterol) who do not respond adequately to diet. Also for the reliefof pruritus associated with partial biliary obstruction. PCSK9inhibitors 1) Monoclonal antibodies: Indicated as an adjunct to diet andAlirocumab (Praluent ®), maximally tolerated statin therapy inEvolocumab (Repatha ®) adults who require additional LDL- 2) Genesilencers: cholesterol (LDL-C) lowering due to ALN-PCSsc (Inclisiran)heterozygous familial hypercholesterolemia 3) Vaccines: AT04A orclinical atherosclerotic cardiovascular disease. MTP inhibitorsLomitapide (Juxtapid ®) Used in homozygous familial hypercholesterolemia(HoFH) patients to reduce low-density lipoprotein cholesterol (LDL-C),total cholesterol (TC), apolipoprotein B (apo B), and non-high- densitylipoprotein cholesterol (non-HDL-C). ApoB antisense Mipomersen(Kynamro ®) Used in patients with homozygous familial oligonucleotideshypercholesterolemia as an adjunct to diet and other lipid-loweringmedications.

Compounds of the present invention may be administered in combinationwith an additional agent or pharmaceutical composition that increasesthe bioavailability or slows the metabolism of the compounds. Agents orpharmaceutical compositions that may increase the bioavailability orslow the metabolism of the compounds herein include inhibitors of atleast one isoform of the cytochrome P450 (CYP450) enzymes, preferablyCYP1A2, CYP2d6, CYP2C9, CYP2C19 and CYP3A4. Suitable agents that may beused to inhibit CYP3A4 include, but are not limited to, nefidipine andritonavir. Such combinations may be administered such that a compound orcompounds of the present invention are present in a single formulationor in the form of separate formulations that may be administeredsequentially with an appropriate period of time in between orsimultaneously. The choice of whether to include the compound orcompounds of the present invention in the same formulation as theadditional agent or agents is within the knowledge of one of ordinaryskill in the art.

Preparation of Intermediates and Compounds

In some embodiments, the first approach (FIG. 1) starts from pyridoxinewhich is modified to produce Intermediate I using methodologiesdescribed in Paul et al. (J. Med. Chem. 1977, 20, 745). Intermediate Imay be modified to produce a protected hydroxamic acid (II) by esterdisplacement. The isopropylidene protecting group of II may be removedby mild hydrolysis with formic acid to give intermediate III. Selectiveoxidation of the 5-CH₂OH group of III ca be performed using manganesedioxide (MnO₂) and III can spontaneously cyclize to the correspondinglactone intermediate (IV). In some embodiments, IV is substituted withan amine, producing the corresponding amide V. Protecting groups can beremoved using hydrogenolysis giving the desired product VI.

In some embodiments, the second approach (FIG. 2) commences withintermediate VIII. VIII can be obtained from Intermediate I, using steps2 and 3 of FIG. 1: removal of the isopropylidene protecting group byhydrolysis (as step 2 of FIG. 1), followed by selective oxidation of5-CH₂OH with MnO₂ (as step 3 of FIG. 1). VIII may be then hydrolyzedusing potassium trimethylsilanoate to generate intermediate VII. VII maybe transformed to intermediate IV using O-protected hydroxylamine andaryl sulfonyl halide. In some embodiments, VI is substituted with anamine, producing the corresponding amide V. The protecting groups can beremoved by hydrogenolysis (VI). In some embodiments, furtherhydrogenation yields the desired product IX.

In some embodiments, the third approach (FIG. 3) starts with theprotected pyro as in FIG. 1 and generates lactol IV by controlledoxidation with MnO₂. The lactol IV can be readily converted to the amineX through reductive amination and further reduction by catalytichydrogenation generating compound XI.

In some embodiments, the fourth approach (FIG. 4) starts from pyridoxineand generates intermediate XIII in a manner similar to that described inAdamczyk M. et al. (Tetrahedron 2000, 56, 2379). XIII can then beoxidized selectively at the 2 methyl group through an N-oxideintermediate XIV followed by rearrangement to the alcohol XV. Furtherstepwise oxidation can yield an aldehyde XVI, followed by an ester XVII.In some embodiments, hydrolysis of the isopropylidene of XVII anddisplacement of the ester XVIII with hydroxylamine yields the desiredproduct compound XIX.

In some embodiments, the fifth approach (FIG. 5), for synthesis of DENVRdRp inhibitor compounds of invention, starts from the intermediate XVIIof FIG. 4. XVII can be hydrolyzed to the acid XX and XX is subsequentlycoupled to O-benzylhydroxylamine to give XXI. XXI may be subjected tohydrogenation, yielding the product compound XXIII, or to hydrolysis,yielding the product compound XXII.

In some embodiments, the sixth approach (FIG. 6) begins from7-(benzyloxy)-6-methylfuro[3,4-c]pyridin-3(1H)-one, intermediate XXIV(Paul, B. et al. J. Het. Chem., 1976, 13, 701). XXIV can be reacted withan amine yielding intermediate XXV. XXV may then be alkylated to XXVIand oxidized selectively at the 2 position through an N-oxiderearrangement, as described in FIG. 4, to yield intermediate XXX. XXXmay then be displaced and hydrogenated to yield the desired productcompound XXXII.

In some embodiments, the seventh approach (FIG. 7) begins from ananalogue of compound XVII of FIG. 5. 17e may be acidified in anhydrousconditions to yield product XXXIII. This product can be oxidized to givethe intermediate aldehyde XXXIV and further oxidized, under controlledconditions, to give the carboxylic acid intermediate XXXV.

In some embodiments, the eighth approach (FIG. 8) consists of reductiveamination of intermediate XXXIV with either a primary or secondaryamines to obtain XXXVI or XXXVII respectively. XXXVII analogues may beobtained by another reductive amination of aldehydes with XXXVI.Treatment of XXXVI and XXXVII analogues with aqueous acids can yield theintermediate XXXVIII, which can be further reacted with hydroxylaminesolutions to give product XXXIX.

In some embodiments, the ninth approach (FIG. 9) begins withintermediate XXXIII, which is reacted in a Mitsunobu reaction withphenols to yield ethers XLIV. These ethers may then be exposed toaqueous formic acid, and then reacted with hydroxylamine to yieldproducts XLV.

In some embodiments, the tenth approach (FIG. 10) consists of acylationof compounds XXXVI by activated carboxylic acids, acyl halides,chloroformates isocyanates, and other electrophiles, to obtain compoundsof the form XLVI. XLVI compounds can then be exposed to aqueous formicacid, and reacted with hydroxylamine to yield product XLVII.

In some embodiments, the eleventh approach (FIG. 11) begins with aWittig reaction of intermediate XXXIV to yield alkenes of the formXLVII. XLVII may then be hydrogenated producing saturated alkanes, thenfurther exposed to aqueous formic acid, and reacted with hydroxylamineto yield products L.

In some embodiments, the twelfth approach (FIG. 12) begins withintermediate XXXIII and reaction of XXXIII with methane sulfonylchloride to yield the reactive intermediate alkyl chloride LI. In someembodiments, LI is immediately and cautiously reacted with a mercaptanto yield the thioether LII. Reaction of LII with excess peroxide mCPBAcan yield a sulfone-N-oxide intermediate LIII, which upon exposure totrifluoroacetic anhydride can rearrange to products LIV. Theintermediate alcohol may then be oxidized in a stepwise manner to LVesters, which are then exposed to aqueous formic acid and reacted withhydroxylamine to yield products LVI.

It can be appreciated by those skilled in the art that the abovesynthetic schemes are not intended to be a comprehensive list of allmeans by which the above compounds may be synthesized. Further methodswill be evident to those of ordinary skill in the art.

General Procedures

General procedures can be found as described in U.S. Pat. No. 8,742,123B2.

EXAMPLES

The following examples are provided to illustrate, but are not limitedto, the presently claimed invention.

Example 1 Preparation of5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide (Compound 1)

Step 1a. Preparation of methyl5-((4-fluorophenyl-amino)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate(Compound 1a): Acetic acid (50 μL, 0.87 mmol) was added to a mixture of5-formyl-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylic acidmethyl ester (J. Org. Chem. 1999, 64, 4537) (220 mg, 0.87 mmol) and4-fluoroaniline (125 μL, 1.3 mmol) in dry methanol (4 mL) and stirred atroom temperature for 15 min and sodium cyanoborohydride (72 mg, 1.3mmol) was added. This mixture was stirred at room temperature for 3 hfollowed by evaporation of 90% of the methanol volume under reducedpressure. The residue was extracted with dichloromethane (3×25 mL) andthe combined organic layers were dried (anh. Na₂SO₄), filtered andconcentrated under reduced pressure. The crude product was purified bysilica gel (methanol/dichloromethane, 0 to 10% methanol) to give 0.290 gof methyl5-((4-fluorophenylamino)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate(Compound 1a) (96%) as a white solid MS-ESI m/z 347 [MI-1]+.

Step 1b. Preparation of5-((4-fluorophenylamino)methyl)-3-hydroxy-4-(hydroxymethyl)picolinate(Compound 1b): Formic acid (2 mL) was added to methyl5(4-fluorophenylamino)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]py-ridine-8-carboxylate(180 mg, 0.5 mmol) at 0° C. and stirred at room temperature for 2 h.Evaporation under reduced pressure afforded a residue5-((4-fluoropheny-lamino)methyl)-3-hydroxy-4-(hydroxymethyl) picolinate(Compound 1b), which was triturated in acetonitrile. MS-ESI m/z 307[MH]+.

Step 1c. Preparation ofN5-(3,4-difluorobenzyl)-N²,3-dihydroxy-4-(hydroxymethyl)pyridine-2,5-dicarboxamide(Compound 1c): Diisopropylethylamine (142 μL, 0.8 mmol) andhydroxylamine hydrochloride (45 mg, 0.64 mmol) were added to a solutionof 5-((4-fluorophenylamino)methyl)-3-hydroxy-4-(hydroxymethyl)picolinate(Compound 1b) (50.0 mg, 0.16 mmol) in methanol (1.0 mL). The reactionmixture was heated to 55° C. for 16 h. The reaction mixture was allowedto cool to room temperature and a saturated solution of ammoniumchloride was added. The reaction mixture was extracted with ethylacetate (3×25 mL) and the combined organic layers were dried (anhydrousNa₂SO₄), filtered and concentrated under reduced pressure to give asolid residue which was recrystallized in acetonitrile to give Compound1.

Example 2 Preparation of5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide (Compound 2)

Step 2a. Preparation of methyl5-((4-fluorophenoxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate(Compound 2a): Triphenylphosphine (0.105 g, 0.40 mmol), followed bydiethyl azodicarboxylate (DEAD) (0.06 mL, 0.40 mmol) were added to asolution of methyl5-(hydroxymethyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate(0.102 g, 0.40 mmol) in 10 mL of THF, at room temperature under Argon(Ar). The resulting mixture was stirred at room temperature for 6 h andthen concentrated. The crude residue was purified by chromatography(SiO₂) with hexanes/ethyl acetate (1:1) as eluent to afford the titlecompound with a contaminant. LC-MS (M+H)⁺ m/z 348.

Step 2b. Preparation of methyl5-((4-fluorophenoxy)methyl)-3-hydroxy-4-(hydroxymethyl)picolinate(Compound 2b): A solution of methyl5-((4-fluorophenoxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate(Compound 2a) (0.129 g, 0.37 mmol) in 3 mL of formic acid was stirred at23° C. for 2 h and then it was concentrated. The crude residue waspurified by chromatography (SiO₂) with hexanes/ethyl acetate (3:7) aseluent to afford the title compound as a white solid: LC-MS (M+H)⁺ m/z308; 1H NMR (DMSO-d₆): 1.39 (s, 6H), 4.80 (s, 2H), 4.89 (s, 2H), 5.01(s, 2H), 7.39-7.46 (m, 55 5H), 8.38 (s, 1H), 10.04 (s, 1H).

Step 2c. Preparation of Compound 2: Hydroxylamine hydrochloride (0.045g, 0.65 mmol) and N,N-diisopropylethylamine (0.14 mL, 0.81 mmol) wereadded to a solution of methyl5-((4-fluorophenoxy)methyl)-3-hydroxy-4-(hydroxymethyl)picolinate(Compound 2b) (0.050 g, 0.16 mmol) in MeOH (3 mL) and heated to 70° C.for 5 h. The crude mixture was diluted with EtOAc and washed withsaturated aqueous ammonium chloride solution and brine. The organicextract was dried (Na₂SO₄), filtered and concentrated in vacuo to affordthe title compound (0.048 g, 96% yield): LC-MS (M+H)⁺ m/z 309.

Example 3 Preparation of5-(3-chloro-4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide (Compound 3)

Step 3a. Preparation of methyl5-((3-chloro-4-fluorophenoxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate(Compound 3a): Using similar procedure as described in the Example 2 ofmethyl 5((4-fluorophenoxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate (Step 2a), obtained compound 3a:ESI-MS (M+H)+ m/z 382.

Step 3b. Preparation of methyl5-((3-chloro-4-fluorophenoxy)methyl)-3-hydroxy-4(hydroxymethyl)picolinate (Compound 3b): Using similar procedure as described inExample 2, in the preparation of methyl5-((4-fluorophenoxy)methyl)-3-hydroxy-4-(hydroxymethyl)picolinate(Compound 2b), Compound 3b was obtained as a white solid (63% yield);LC-MS (M+H)⁺ m/z 342.

Step 3c. Preparation of Compound 3: Using similar procedure as describedin the Example 2, in the preparation of5-((4-fluorophenoxy)methyl)-N,³-dihydroxy-4-(hydroxymethyl)picolinamide,Step 2c, Compound 3 was obtained as a beige solid (98% yield); LC-MS(M+H)⁺ m/z 342.

Example 4 Preparation ofN⁵-(3-chloro-4-fluorobenzyl)-N²,3-dihydroxy-4-(hydroxymethyl)pyridine-2,5-dicarboxamide(Compound 4)

N²,3-bis(benzyloxy)-N⁵-(3-chloro-4-fluorobenzyl)-4-(hydroxymethyl)pyridine-2,5-dicarboxamide(0.03 g, 0.330 mmol) and 10% Pd/C catalyst (25 mg) were stirred inmethanol (4.0 mL) under an atmosphere of hydrogen for 1 h. The catalystwas filtered and the reaction mixture was concentrated under vacuumyielding 0.015 g ofN⁵-(3-chloro-4-fluorobenzyl)N²,3-dihydroxy-4-(hydroxymethyl)pyridine-2,5-dicarboxamideas a white solid; ¹H NMR (400 MHz, DMSO-d₆, ppm): δ 13.01 (s, 1H), 12.0(s, 1H), 9.51 (s 1H), 9.02 (t 1H), 8.11 (s, 1H), 7.60 (d 1H), 7.40 (m,2H), 4.68 (s, 2H), 4.48 (s, 2H); MS-ESI m/z 370 [MH]⁺.

Example 5 Preparation of 5-{(1-3-chloro-4-fluoro-phenyl)aminomethyl}2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylic acidhydroxamide (Compound 5)

251 mg of5-formyl-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylic acidmethyl ester (J. Org. Chem. 1999, 64, 4537) (1 mmol) and3-chloro-4-fluorobenzylamine (125 μL, 1.3 mmol) in dry methanol (4 mL)were stirred at room temperature for 15 min and sodium cyanoborohydride(72 mg, 1.3 mmol) was added. This mixture was stirred at roomtemperature for 3 h followed by evaporation of 90% of the methanolvolume under reduced pressure. The residue was extracted withdichloromethane (3×25 mL) and the combined organic layers were dried(anh. Na₂SO₄), filtered and concentrated under reduced pressure. Thecrude product was purified by silica gel (methanol/dichloromethane, 0 to10% methanol) to give 0.300 mg of5-{(1-3-chloro-4-fluoro-phenyl)aminomethyl}2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylic acidhydroxamide. 50 mg of this compound was then dissolved in dry EtoAc anda solution of HCl in ether was added dropwise until precipitate was nolonger seen to form. This precipitate was filtered and dried in adesiccator yielding 40 mg of a slightly hygroscopic material.

Example 6 Preparation of5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxamide (Compound 6)

Compound 6 was synthesized by reaction of 5-formyl (0.751 g, 3 mmol) and1000 mg (3.5 mmol) (4-fluorobenzyl) triphenyphosphonium bromide in THFat −78° C. then stirred overnight at room temperature. The reaction wasquenched with NH₄Cl (saturated). The crude product was separated onsilica gel to yield 417 mg (31% yield). This product was hydrogenated bycatalytic hydrogenation over Pd/C 5% in EtOAc. Silica gel chromatographypurification yielded 305 mg, which was reacted with hydroxylamine(excess) in ethanol (2 h, 80° C.). The compound was precipitated 1N HCl,filtered and redissolved in MeOH with the addition of 6 N HCl, stirredfor 4 h, followed by evaporation; providing the desired product. MS-ESIm/z 307 [MH]⁺.

Example 7 Preparation of5-(4-methoxy-benzyloxymethyl)-3-hydroxy-4-methyl-pyridine-2-carboxylicacid (Compound 7)

0.05 g (0.15 mmol) of Compound 8 was dissolved in dry THF. 2 equiv (0.3mmol) of potassium trimethylsilanoate was added and the mixture warmedforming a slurry. Rapid stirring overnight at 75° C. showed completeconversion. Concentration of the solution, resolvation in 25 mL EtoAcand acidification with 1.0 N HCl was done with the extraction of thecompound into the organic phase. The organic phase was dried over brinethen evaporated to dryness giving the desired compound.

Example 8 Preparation of5-(4-methoxy-benzyloxymethyl)-3-hydroxy-4-methyl-pyridine-2-carboxylicacid methyl ester (Compound 8)

0.5 g (1.5 mmol) of methyl3-hydroxy-4-(hydroxymethyl)-5-((4-ethoxybenzyloxy)methyl)picolinate wasdissolved in 2 mL Ac₂O and stirred for 2 h. 5 mL MeOH was added and thesolvent was evaporated to dryness. The residue was dissolved in 50 mLdegassed, N₂ flushed EtOAc. 0.25 g 10% Pd/C was added. H₂ gas wasbubbled in for 4 h and the mixture left to stir overnight. N₂ wasbubbled in and the solution filtered over a pad of silica gel. The clearsolution was then evaporated to dryness and the resulting syrupcrystallized to give the desired product quantitatively.

Example 9 Preparation of5-((4-methoxybenzyloxy)methyl)-2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine(Compound 9)

Anhydrous THF (200 mL) was added to NaH (60%, 24 g, 600 mmol) at 0° C.under a nitrogen atmosphere. To this suspended mixture a solution of2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)methanol (32.0 g, 150mmol) in 400 mL of THF (J. Med. Chem., 1977, 20, 745) was added. Theresulting mixture was refluxed for 30 min; a significant amount ofprecipitate accumulated during the reflux. After cooling to roomtemperature, p-methoxybenzyl chloride (23.5 g, 150 mmol) was introduceddrop-wise and the resulting mixture was refluxed for another 8 h. Thereaction was quenched carefully by adding ice-cold water to the viscousmixture at 0° C. and diluted with a saturated ammonium chloride solutionfollowed by extraction with methylene chloride. The combined organicextracts were washed with brine, dried (Na₂SO₄), and concentratedyielding a brown oil. The crude product was purified by chromatography(10% ethyl acetate/petroleum ether) yielding 25.0 g of Compound 9 (50%yield); LC-MS (M+H)+ m/z 331.

Example 10 Preparation of methyl5-((4-methoxybenzyloxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate (Compound 10)

Step 10a.5-((4-Methoxybenzyloxy)methyl)-2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine7-oxide

5-((4-Methoxybenzyloxy)methyl)-2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine(50.0 g, 152 mmol, 1.0 equiv) was dissolved in dry CH₂Cl₂ (500 mL) andthe solution was cooled to 0° C. To this solution was addedm-chloroperbenzoic acid (85% purity of the reagent, 37.0 g, 182 mmol,1.2 equiv). After being stirred at 23° C. for 12 h, the reaction mixturewas extracted with Na₂SO₃(10%, 2×200 mL), NaHCO₃ (5%, 2×200 mL), H₂O,dried (Na₂SO₄), filtered and the solvent was removed under reducedpressure. The crude product was purified by chromatography (10%methanol/ethyl acetate) to afford the title compound as pale-yellowsolid (35 g, 68%); LC-MS (M+H)⁺ m/z 346.

Step 10b.(5-((4-Methoxybenzyloxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridin-8-yl)methanol

Trifluoroacetic anhydride (4.5 mL, 32 mmol) was added to a solution of5-((4-methoxybenzyloxy)methyl)-2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridine7-oxide (21.2 g, 61 mmol) in dry CH₂Cl₂ (200 mL) at 0° C. and stirredfor 5 min. An additional amount of trifluoroacetic anhydride (11.5 mL,82.7 mmol) was added and the reaction mixture was stirred overnight at23° C. Then, the reaction mixture was cooled to 0° C. and MeOH (150 mL)was added while stirring was continued. The solvents were evaporated andthe resulting residue was dissolved in CH₂Cl₂ and washed with Na₂CO₃(20% aqueous) and H₂O until pH was neutral. The organic phase was dried(Na₂SO₄), filtered and concentrated in vacuo. The residue wascrystallized from EtOH—CH₂Cl₂ to afford the title compound (17.5 g,83%); LC-MS (M+H)⁺ m/z 346.

Step 10c.5-((4-Methoxybenzyloxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carbaldehyde

To a solution of(5-((4-methoxybenzyloxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridin-8-yl)methanol(14.60 g, 42.3 mmol) in ethyl acetate (500 mL) was added IBX (35.5 g,128 mmol) and the suspension was heated to reflux for 4 h. Theprecipitate was removed by filtration and the filtrate was concentratedunder reduced pressure to afford the title compound (14.0 g, 95%). Thecrude material was used for the next step without further purification:LC-MS (M+H)⁺ m/z 344.

Step 10d. Methyl5-((4-methoxybenzyloxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate

To the solution of5-((4-methoxybenzyloxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carbaldehyde(10.3 g, 30 mmol) in anhydrous MeOH (120 mL), KOH (85%, 5.3 g, 78 mmol)and iodine (9.9 g, 39 mmol) were added at 0° C. The reaction mixture waskept at 23° C. and stirred for 12 h until no starting material wasdetected by TLC. Then, the solution was treated with Na₂SO₃ (solid) andthe pH was adjusted to 7. The solid was filtered and solvent was removedunder reduced pressure. The residue was dissolved in EtOAc and washedwith water. The combined organic extracts were dried (Na₂SO₄), filteredand concentrated in vacuo. The crude residue was purified bychromatography (SiO₂) with petroleum ether:ethyl acetate (5:1) as eluentto afford the title compound as pale-yellow solid (8.8 g, 78%): LC-MS(M+H)⁺ m/z 374.

Example 11 Preparation of5-(4-methoxybenzyloxymethyl-3-hydroxy-4-methyl-pyridine-2-carboxylicacid hydroxyamide (Compound 11)

Step 11a. Methyl3-hydroxy-4-(hydroxymethyl)-5-((4-methoxybenzyloxy)methyl)picolinate

The solution of methyl5-((4-methoxybenzyloxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate(8.8 g, 23.6 mmol) in 200 mL of HCl/MeOH was stirred at 23° C. for 24 h.MeOH (500 mL) was added to dissolve the suspension and NaHCO₃ (solid)was added to neutralize the reaction mixture. The excess solid wasfiltered and the solvent was removed under reduced pressure. The residuewas dissolved in EtOAc and washed with water. The combined organicextracts were dried (Na₂SO₄), filtered and concentrated in vacuo toafford the title compound as a light yellow solid (6.0 g, 100%): LC-MS(M+H)+ m/z 334.

Step 11b.N,3-Dihydroxy-4-(hydroxymethyl)-5-((4-methoxybenzyloxy)methyl)picolinamide

To a solution of methyl3-hydroxy-4-(hydroxymethyl)-5-((4-methoxybenzyloxy)methyl)picolinate(0.050 g, 0.15 mmol) in MeOH (3 mL) was added hydroxylaminehydrochloride (0.042 g, 0.60 mmol) and N,N-diisopropylethylamine (0.13mL, 0.75 mmol). The resulting mixture was placed in the microwave andheated to 80° C. for 1.5 h. The crude mixture was diluted with EtOAc andwashed with saturated aqueous ammonium chloride solution and brine. Theorganic extract was dried (Na₂SO₄), filtered and concentrated in vacuoto afford the title compound (0.039 g, 78%): LC-MS (M+H)⁺ m/z 335.

Example 12 Preparation of methyl5-((4-fluorophenylamino)methyl)-3-hydroxy-4-(hydroxymethyl)picolinate(Compound 12)

Formic acid (2 mL) was added to methyl5(4-fluorophenylamino)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate(180 mg, 0.5 mmol) at 0° C. and stirred at room temperature for 2 h.Evaporation under reduced pressure afforded a residue5-((4-fluorophenylamino)methyl)-3-hydroxy-4-(hydroxymethyl) picolinate,which was triturated in acetonitrile. MS-ESI m/z 307 [MH]⁺.

Example 13 Preparation ofN⁵-(2-N-methylcarboxamide-4-fluorobenzyl)-N²,3-hydroxy-4-(hydroxymethyl)pyridine-2-methylcarboxylate,5-carboxamide (Compound 13)

100 mg of5-carboxyl-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylic acidmethyl ester was dissolved in 2 mL DMF and 0.2 mL N-methylmorpholine wasadded with about 300 mg HBTU. 100 mg2-N-methylcarboxamide-4-fluorobenzylamine was added to the solutionafter approximately 30 min. The mixture was stirred overnight afterwhich it was quenched by addition to 20 mL 10% citric acid. 25 mL EtOAcwas added and the organic phase was washed with citric acid (2×), K₂CO₃(2×), and brine. Yield was 101 mg. LCMS (100%) MS-ESI m/z 432[MH]+.

Example 14 Preparation of methyl3-(benzyloxy)-5-((4-fluorophenoxy)methyl)-4-methylpicolinate (Compound14)

Using a procedure similar to that described in the Example 2, step 2a,Compound 14 was prepared as a colorless oil: 72% yield. LC-MS (M+H)F m/z382.

Example 15 Preparation of methyl5-((4-fluorophenoxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate(Compound 15)

Triphenylphosphine (0.105 g, 0.40 mmol), followed by diethylazodicarboxylate (DEAD) (0.06 mL, 0.40 mmol) were added to a solution ofmethyl5-(hydroxymethyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate(0.102 g, 0.40 mmol) in 10 mL of THF, at room temperature under Ar. Theresulting mixture was stirred at room temperature for 6 h and thenconcentrated. The crude residue was purified by chromatography (SiO₂)with hexanes/ethyl acetate (1:1) as eluent to afford the title compoundwith a contaminant. LC-MS (M+H)+ m/z 348.

Example 16 Preparation of methyl5-((4-fluorophenoxy)methyl)-3-hydroxy-4-(hydroxymethyl) picolinate(Compound 16)

A solution of methyl5-((4-fluorophenoxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate(0.129 g, 0.37 mmol) in 3 mL of formic acid was stirred at 23° C. for 2h and then it was concentrated. The crude residue was purified bychromatography (SiO₂) with hexanes/ethyl acetate (3:7) as eluent toafford the title compound as a white solid. LC-MS (M+H)+ m/z 308; ¹H NMR(ppm, DMSO-d₆): 1.39 (s, 6H), 4.80 (s, 2H), 4.89 (s, 2H), 5.01 (s, 2H),7.39-7.46 (m, 55 5H), 8.38 (s, 1H), 10.04 (s, 1H).

The solid was then dissolved in THF containing 1 mmol KOTMS and refluxedfor 1 h. The resulting slurry was dropped in cold hexanes and filteredoff. The hygroscopic filtrated was dissolved in water and acidified with90% formic acid to form the carboxylic acid. This was filtered off anddried in vacuo. Yield: 40 mg off-white powder (blue fluorescence underUV).

Example 17 Preparation of methyl5-((4-fluorophenoxy)methyl)-3-hydroxy-4-methylpicolinate (Compound 17)

A solution of methyl3-(benzyloxy)-5((4-fluorophenoxy)methyl)-4-methylpicolinate (0.245 g,0.64 mmol) in ethyl acetate (10 mL) was hydrogenated under 1 atm ofhydrogen at 23° C. over 10% palladium on activated carbon for 1 h. Thereaction mixture was filtrated and the solution was concentrated invacuo to afford Compound 17 as a white solid (0.171 g, 92%). LC-MS(M+H)+ m/z 292.

Example 18 Preparation of(3-chloro-4-fluoro-benzyl)-(5-hydroxy-6-hydroxycarbamoyl-4-hydroxymethyl-pyridin-3-ylmethyl)-carbamicacid benzyl ester (Compound 18)

70 mg of Compound 5 was dissolved in DCM (20 mL). Triethylamine (100 μL)was then added followed by benzylchloroformate (˜60 mg). The mixture wasstirred for 4 h after which 20 mL 10% citric acid was added. The DCMphase was removed and further washed with K₂CO₃, dried over Mg₂SO₄ andevaporated to dryness. 1 mL formic acid (90%) was added and the solutionwarmed to 75° C. for 4 h. The solution was diluted with water forming aprecipitate which was filtered and dried. The precipitate was taken upin 1 mL pyridine and 250 μL of 50% aq. hydroxylamine was added. Theyellow solution was warmed to 80° C. for 8 h, after which the pyridinewas removed in vacuo. Careful addition of cold 5% acetic acid yielded aprecipitate which was filtered and washed with cold 5% acetic acid.Yield: 51mg product. LC-MS (M+H)^(F) m/z 490.1.

Example 19 Preparation of4-hydroxymethyl-3-methoxy-pyridine-2,5-dicarboxylic acid5-(4-fluoro-benzylamide) 2-hydroxyamide (Compound 19)

3-hydroxy-4-hydroxymethyl-pyridine-2,5-dicarboxylic acid5-cyclohexylmethyl-amide 2-hydroxyamide (0.060 g, 0.137 mmol, 1 equiv)and 10% Pd/C (5 mg) in methanol (4.0 mL) were stirred under anatmosphere of hydrogen 1 h. The catalyst was filtered and reactionmixture was concentrated under vacuum to give 0.040 g of 53 (85%) as awhite solid. MS-ESI m/z 350 [MH]+.

Example 20 Preparation of3-hydroxy-4-hydroxymethyl-pyridine-2,5-dicarboxylic acid 5-dibenzylamide2-hydroxyamide (Compound 20)

Procedure as described in U.S. Pat. No. 8,742,123 B2 Example 55.

Example 21 Preparation of5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide (Compound 21)

550 mg of Compound I, of FIG. 1, was dissolved in 30 mL DCM was reactedwith 2.5 equiv of methane sulfonyl chloride in the presence of 5 equivtriethylamine. Extraction was done with 5% citric acid, drying withNa₂SO₄, and evaporation yielded 500 mg of desired mesylate. Thismesylate was immediately reacted with 400 mg of benzene sulfinic acid inDMF (2 mL). The product was isolated by precipitation in water andfiltration. The crude was dissolved in CHCl₃ (30 mL) and 400 mg mCPBA(70%) was added. After 1 h stirring, the reaction was extracted usingK₂CO₃ and the organic phase dried over CaCO₃ the evaporated. The residuewas dissolved in 3 mL DCM and 3 mL trifluoroactetic anhydride was added.Stirring at reflux 45° C. for 20 h affording the rearranged product,isolated through evaporation of solvent. The residue was then added to asolution of MnO₂ 2 g in CHCl₃ (30 mL) and stirred at reflux 1 h.Filtration and evaporation afforded the aldehyde (250 mg). This wasplaced in 10 mL MeOH with 1.2 equiv I₂ and 3 equiv KOTMS. Stirring atroom temperature for 1 h gave the ester in quantitative conversion. Theproduct was purified on silica gel. 100 mg of the ester was reacted withexcess (hydroxylamine 50% aquiv) in pyridine to give the hydroxamate.Dilution in EtOAc and extraction with 5% citric acid gave the desiredintermediate. The final product was obtained by adding 50 mg of theabove acetonide to neat 70% formic acid. After 15 min the reaction iscomplete, the formic acid is evaporated off and the residue trituratedwith water to give Compound 21 as a white powder. MS-ESI m/z 339 [MH]⁺.

Example 22 Preparation of5-((3-chloro4-fluorobenzylamino)methyl)-4-(hydroxymethyl)picolinhydroxamide(Compound 22)

25 mg of Compound 18 and 10% Pd/C catalyst (5 mg) were stirred in 4 mLof methanol under an atmosphere of hydrogen for 12 h. The catalyst wasfiltered and reaction mixture was concentrated under vacuum yielding0.018 g of Compound 22 as a white solid; MS-ESI m/z 356 [MH]+.

Example 23 Preparation of3-hydroxy-4-hydroxymethyl-pyridine-2,5-dicarboxylic acid5-(4-fluoro-benzylamide)2-(methoxy-amide) (Compound 23)

Formic acid (2 mL) was added to2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-5,8-dicarboxylic acid5-(4-fluoro-benzylamide)8-(methoxy-amide) (0.048 g, 0.123 mmol,1 equiv).The reaction mixture was stirred at room temperature 10 min. Formic acidwas concentrated under vacuum and solid was triturated with diethylether under to give 0.030 g of Compound 23 (71%) as a white solid.MS-ESI m/z 350 [MH]+.

Example 24 Preparation of5-(4-fluoro-benzyloxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide (Compound 24)

Procedure as described in U.S. Pat. No. 8,742,123 B2, Example 34, or asin Compound 11, using 4-fluorobenzyl bromide in the initial step.

Example 25 Preparation of2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-5,8-dicarboxylic acid5-(3-chloro-4-fluoro-benzylamide)-8-(methyl ester) (Compound 25)

Prepared as in Example 13a using 3-chloro-4fluorobenzyl amine in theinitial step.

Example 26 Preparation of5-((4-fluorophenoxy)methyl)-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]picoline(Compound 26)

2.09 g (3.5 mmol) of5-hydroxymethyl2,2-dimethyl-4H-[1,3]dioxino[4,5-c]picoline was dissolvedin 50 mL EtOAc then cooled to 0° C. 1 mL triethylamine was addedfollowed by 1.15 g pf methane sulfonyl chloride. The mixture was stirredfor 30 min. A solution of 1.12 g 4-fluorophenol in 5 mL DMF was cooledto 0° C. and 1.12 g potassium tert-butanoate was added to form a phenolsalt. This solution was added to the other at which point a gel formed.This was stirred and allowed to warm to room temperature for 1 h. Thesolution was then quenched by addition of water and adjusting the pH to4 with acetic acid. The organic phase was evaporated. The residue wasthen adsorbed on silica gel then washed over a pad of silica gel withDCM. The DCM was evaporated to yield 2.4 g of a clear oil whichspontaneously crystallized.

Example 27 Preparation of5-((4-fluorophenoxy)methyl)-3-hydroxy-4-(hydroxymethyl)picoline(Compound 27)

50 mg of the compound from Example 26 was dissolved in 1 mL of 90%formic acid. The mixture was warmed to 75° C. for 4 h. The formic acidwas removed by vaccum and the residue triturated in water to give anoff-white powder suspension. This was filtered to give 26 mg of thedesired compound.

Example 28 Cell Culture and Treatments

Human hepatoma cell lines HepG2 and Huh-7 were routinely cultivated inDulbecco's modified Eagle's medium (DMEM; catalog no. 319-005-CL,Wisent) supplemented with 10% fetal bovine serum (FBS; catalog no.080-350, Wisent). For phenotypic screening of LDLR small moleculeinducers Examples 1-27, cells were incubated overnight in conditionedmedia containing giving chemical compounds 1-27 resuspended in DMSO atconcentration ranging from 100 μM to 10 μM. For sterol-regulatedconditions, HepG2 cells were incubated in 5% LPDS, 50 μM mevastatin, and50 μM mevalonolactone in the absence (-sterols) or presence of 1 μg/ml25-hydroxycholesterol and 10 μg/ml cholesterol (+sterols). For inhibitortreatments HepG2 cells were treated with DMSO or 10 μM compounds 2 incombination with U0126 (25 μM, catalog no. 9903, Cell SignalingTechnology), Cycloheximide (CHX; 20 μg/ml, catalog no. C7698,Sigma-Aldrich), PD153035 (20 μM, catalog no. 14879, Cayman Chemical),bisindolylmaleimide I (BIM; 10 μM, catalog no. 13298, Cayman Chemical),H-89 (10 μM, catalog no. 10010556, Cayman Chemical), Dorsomorphin (5 μM,catalog no. 11967, Cayman Chemical) or 5-Azacytidine (10 μM, catalog no.A2385, Sigma-Aldrich). FIG. 19 shows that CHX and U0126, and no othertested inhibitor, block increase of LDLR by Compound 2.

Human embryonic kidney 293 (HEK-293, catalog no. CRL-1573; ATCC) cellswere cultivated in complete DMEM without sodium pyruvate (catalog no.319-015-CL, Wisent) and human umbilical vein/vascular endothelium cells(HUVEC; catalog no CRL-1730, ATCC) in F12 K media (catalog no.312-250-CL, Wisent) both supplemented with 10% fetal bovine serum (FBS;catalog no. 080-350, Wisent).

Example 29 Western Blot Analysis

Treated cells from Example 28 were washed three times inphosphate-buffered saline (PBS) and lysed in radioimmune precipitationassay buffer (50 mM Tris/HCl, pH 8.0, 1% (v/v) Nonidet P-40, 0.5% sodiumdeoxycholate, 150 mM NaCI, and 0.1% (v/v) SDS) supplemented with acomplete protease inhibitor mixture (catalog no. 11 697 498 001, RocheApplied Science). Proteins were separated by 8% SDS polyacrylamide gelelectrophoresis, blotted on nitrocellulose membranes (Bio-Rad), andblocked for 1 h in Tris-buffered saline-Tween 20 (TBS-T; 50 mM Tris-HCl,pH 7.5, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat dry milk.Membranes were then incubated overnight in TBS-T supplemented with 1%nonfat milk and the indicated antibodies; goat anti-human LDLR (1:1000;catalog no. AF2148, R&D Systems), rabbit anti-actin (1:5000; catalog no.A2066, Sigma-Aldrich). Appropriate HRP-conjugated secondary antibodies(1:10,000; GE healthcare) were used for detection using the WesternLightning Ultra chemiluminescence kit (catalog no. NEI112001EA,PerkinElmer Life Sciences) and BioFlex EC Films (catalog no. CLEC810,InterScience). Unsaturated films were numerized using the highresolution CanoScan 9000F scanner and corresponding bands werequantified using the ImageJ software (NIH). Relative quantification ofLDLR protein levels were normalized to that of actin and compared tovehicle (DMSO) herein determined as 1. Results are described in Table 2and illustrated in FIG. 13A to FIG. 13C.

TABLE 2 Result of compound efficacy and cytotoxicity CC50 LDLR Fold LDLRFold Ex. (μM) Change Change No. Chemical Name (MTT) 10 μM 10 nM 15-[(4-fluoro-phenylamino)-methyl]-3- 120 6.9hydroxy-4-hydroxymethyl-pyridine-2- carboxylic acid hydroxyamide 25-(4-fluoro-phenoxymethyl)-3-hydroxy- 50 6.3 24-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide 35-(3-chloro-4-fluoro-phenoxymethyl)- 11 5.63-hydroxy-4-hydroxymethyl-pyridine-2- carboxylic acid hydroxyamide 4N⁵-(3-chloro-4-fluorobenzyl)-N²,3- 2.7dihydroxy-4-(hydroxymethyl)pyridine- 2,5-dicarboxamide 55-{(1-3-chloro-4-fluoro- 2.4 phenyl)aminomethyl}-2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8 carboxylic acid hydroxamide 65-[2-(4-fluoro-phenyl)-ethyl]-3- 39 2.3 1.5hydroxy-4-hydroxymethyl-pyridine-2- carboxylic acid hydroxyamide 75-(4-methoxy-benzyloxymethyl)-3- 1.9 1.7hydroxy-4-methyl-pyridine-2-carboxylic 2-3 acid 85-(4-Methoxy-benzyloxymethyl)-3- 1.7 1.7 hydroxy-4-methyl-pyridine-2-carboxylic acid methyl ester 9 Methyl 5-((4-methoxybenzyloxy)methyl)-1.4 2,2-dimethyl-4H-[1,3] dioxino[4,5- c]pyridine-8-carboxylate 105-((4-methoxybenzyloxy)methyl)-2,2,8- 1.2 1.7 trimethyl-4H-[1,3]dioxino[4,5- c]pyridine 11 5-(4-methoxybenzyloxymethyl-3-hydroxy- 2-34-methyl-pyridine-2-carboxylic acid hydroxyamide 125-((4-fluorophenylamino)methyl)-3- hydroxy-4-(hydroxymethyl)picolinate13 5-(4-fluoro-benzyloxymethyl)-3- hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid methoxy-amide 14 Methyl 5-((4-fluorophenoxy)methyl)-3-hydroxy-4-methylpicolinate 15 Methyl 5-((4-fluorophenoxy)methyl)-3-hydroxy-4-(hydroxymethyl) picolinic acid 16 Methyl5-((4-fluorophenoxy)methyl)- 2,2-dimethyl-4H-[1,3]dioxino[4,5-c]pyridine-8-carboxylate 17 Methyl 3-(benzyloxy)-5-((4-fluorophenoxy)methyl)-4- methylpicolinate 18(3-chloro-4-fluoro-benzyl)-(5-hydroxy-6-hydroxycarbamoyl-4-hydroxymethyl- pyridin-3-ylmethyl)-carbamic acidbenzyl ester 19 4- hydroxymethyl-3-methoxy-pyridine- 6.62,5-dicarboxylic acid 5-(4-fluoro- benzylamide) 2-hydroxyamide 205-benzenesulfonylmethyl-3-hydroxy-4- hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide 21 3-hydroxy-4-hydroxymethyl-pyridine-2,5-dicarboxylic acid 5- dibenzylamide-2-hydroxyamide 22N,3-bis(benzyloxy)-5-((3-chloro4- fluorobenzylamino)methyl)-4-(hydroxymethyl) picolinamide 23 3-hydroxy-4-hydroxymethyl-pyridine-2,5-dicarboxylic acid 5-(4-fluoro- benzylamide)2-(methoxy-amide) 245-(4-fluoro-benzyloxymethyl)-3- hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide 25 2,2-dimethyl-4H-[1,3]dioxino[4,5- 1.9c]pyridine-5,8-dicarboxylic acid 5- (3-chloro4-fluoro-benzylamide)-8-(methyl ester) 26 5-((4-fluorophenoxy)methyl)-2,2- 1.5 1dimethyl-4H-[1,3]dioxino[4,5-c]picoline 275-((4-fluorophenoxy)methyl)-3-hydroxy- 1.2 0.6 4-(hydroxymethyl)picoline

FIG. 15 shows the effects of Compound 2 on LDLR protein levels byWestern blot analyses in HepG2, Huh7, HEK293, and HUVEC cells.

Example 30 Immunocytochemistry

Cells were permeabilized or not with 0.1% Triton X-100/PBS for 10 min,and incubated with 150 mM glycine (to stabilize the aldehydes),incubated for 30 min with 1% BSA (Fraction V, Sigma) containing 0.1%Triton X-100 or not, followed by overnight incubation at 4° C. with goatanti-human LDLR (1:100; R&D Systems, catalog no. AF2148). Afterward,cells were incubated for 60 min with corresponding AlexaFluor-conjugated secondary antibodies (Molecular Probes) and mounted in90% glycerol containing 5% 1,4-diazabicyclo[2.2.2]octane (Sigma).Immunofluorescence analyses were performed with an Olympus FluoViewFV10i confocal microscope. The visualization of increased expression ofthe Low density lipoprotein receptor can be seen in FIG. 14.

Example 31 Reverse Transcription and Quantitative Real-Time PCR

The integrity of total RNA samples, isolated using Ribozol RNAExtraction Reagent (catalog no. N580-100, Amresco), was verified byagarose gel electrophoresis or using an Agilent 2100 BioAnalyzer. cDNAwas prepared using iScript cDNA Synthesis Kit according themanufacturer's instructions (catalog no. 1708891, Bio-Rad Laboratories).Quantitative Real-Time PCR was performed in a MX3000p real-time thermalcycler (Agilent) using the iTaq Universal SYBR Green Supermix, (catalogno. 1725121, Bio-Rad Laboratories). For each gene of interest,dissociation curves and agarose gel electrophoresis were performed toensure unique PCR product. Arbitrary units were determined from PCRduplicates for each sample using the Homo sapiens TATA-box bindingprotein (hTBP) as a normalizer. Oligonucleotides sequences used were:Homo sapiens low-density lipoprotein receptor (hLDLR; SEQ ID NO 1:5′-AGGAGACGTGCTTGTCTGTC, SEQ ID NO 2: 5′-CTGAGCCGTTGTCGCAGT), Homosapiens 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (hHMGCR; SEQ IDNO 3: 5′-GTCACATGATTCACAACAGG, SEQ ID NO 4: 5′-GTCCTTTAGAACCCAATGC),Homo sapiens glyceraldehyde-3-phosphate dehydrogenase (GAPDH; SEQ ID NO5: 5′-GGTGTGAACCATGAGAAGTATGA, SEQ ID NO 6: 5′-GAGTCCTTCCACGATACCAAAG.Homo sapiens proprotein convertase subtilisin-kexin 9 (hPCSK9) mRNAexpression was evaluated on total cDNA using the TaqMan gene expressionassay Hs00545399_m1 (catalog no. 4331182, ThermoFisher Scientific) andthe TaqMan Fast Advanced Master Mix (catalog no. 4444557, ThermoFisherScientific). Results of QPCR LDLR mRNA increase is illustrated in FIG.16.

Example 32 Cell Viability Assay

Cells were seeded into 96-well plates and incubated at confluencyovernight with 1, 5, 10, 50, 100 or 500 μM of given compounds in phenolred-free conditioned media (catalog no. 319-051-CL, Wisent). Followingincubation, media was replaced and cells incubated for 4 h with 100 μLof phenol red-free DMEM containing 0.5 mg/mL of MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolylblue; catalog no. M5655, Sigma-Aldrich). Afterwards, media was removedand converted dye solubilized with 100 μL acidic isopropanol (0.06 N)and absorbance measured at a wavelength of 570 nm with backgroundsubtraction at 650 nm. Arbitrary units of cell viability of a givenexample were determined relative to equal volume of DMSO (vehicle) foreach concentration used. Results are shown in Table 2.

Example 33 Gaussia Luciferase Assay

Human LDLR (−1020 bp) proximal promoter and partial 3′UTR (nt11131317-11132220 chromosome 9) were generated by PCR using genomic DNAfrom HepG2 cells as template and subcloned into pCMV-GLuc vector(catalog no. N8081S, New England Biolabs) in order to replace the CMVpromoter or the 3′UTR region of the vector. Selected clones wereverified by DNA sequencing. Oligonucleotides used were: hLDLR promoter(SEQ ID NO 7: 5′-TGTACTAGTCTTATTCCTGGGGGAACCGC, SEQ ID NO 8:5′-GCAAAGCTTGCTCG-CAGCCTCTG-CCAGGCAGTG and hLDLR 3′UTR (SEQ ID NO 9:5′-TACGCGGCCGCACATCTGCCTGGAGTCCCGTC, SEQ ID NO 10:5′-ACTTCTAGACCCATCCC-AACACACACGACAG). For transient experiments, HepG2cells were seeded in 24-well plates at a density of 1.5 10⁵/well. 24 hlater, cells were transfected in duplicate with the corresponding pGLucconstruct using Lipofectamine 3000 (catalog no. L3000008, Thermo FisherScientific). After overnight incubation, cells were washed twice withDMEM and incubated in 0.5 mL of DMEM without or with given examples atvarious concentration for 24 h. 20 μL of conditioned media was loadedinto black 96-well plates, and relative activity of secreted Gaussialuciferase was assessed by luminescence measurements using the BioLuxkit (catalog no. E3300L, New England Biolabs) and the BioTek Synergy 2microplate reader. High quality of HepG2 cells stability expressingpGLuc-LDLR-3′UTR were also generated following selection with 0.75 mg/mLG418 Sulfate antibiotic (catalog. 400-130-IG, Wisent). Results aredisplayed in FIG. 17.

Example 34 Measurements of mRNA Decay

HepG2 cells were incubated with DMSO or 10 μM of Compound 2 for 24 h.Following incubation, cells were washed twice with DMEM and incubatedwith 5 μg/ml actinomycin D for 0, 60, 120 and 240 min. LDLR and GAPDHrelative mRNA levels were analyzed by Quantitative Real-Time PCR asdescribed in Example 31. Results of QPCR LDLR mRNA stabilization isillustrated in FIG. 18.

Example 35 Microsomal Stability Assay

Pooled human liver microsomes (HLM; Cat #X008067 lot IHG) and pooledmale Sprague Dawley rat liver microsomes (RLM; Cat #M00001 Lot NNK) werepurchased from BioreclamationIVT, Baltimore, Md. NADPH (Cat #N1630, 95%HPLC) and DMSO (GC grade) were purchased from Sigma-Aldrich, Canada.Potassium phosphate buffer (100 mM) with magnesium chloride pH 7.4 wasprepared freshly. Potassium phosphate buffer (100 mM, pH 7.4, +2 mMMgCl2) containing human or rat liver microsomes (0.50 mg/ml) waspre-incubated separately with Compound 2 (1 μM) or positive control(loperamide, 1 μM) in a water bath with the temperature set at 37° C.for 5 min (N=2). Reactions were initiated by adding NADPH (finalconcentration 1 mM) in all the wells. Reactions without NADPH were alsoincubated (final point only) to rule out non-NADPH metabolism orchemical instability in the incubation buffer. Reactions were terminatedat each time point (0, 5, 10, 20, 30, 45 and 60 min) by transferring 50μL in a new 96 well plate and adding 100 μl of acetonitrile containingan internal standard (labetalol at 2 μM). The plates were centrifuged at4000 rpm for 5 min, and an aliquot of supernatant was diluted with onevolume of water+0.1% formic acid before analysis by LC-MS/MS. Referencesamples were also prepared at concentrations between 0.002 and 2 μM inbuffer containing microsomes, by adding the diluted solutions of thecompound to the buffer containing microsomes quenched with 2 volume ofacetonitrile containing the internal standard (whereas the incubation T0is obtained by quenching the microsomes containing the compound, thenadding NADPH). Samples were monitored for parent compound disappearancein Multi Reaction Monitoring (MRM) mode using LC-MS/MS. The peak arearatios of analyte versus internal standard compared to the calibrationcurve were used to calculate concentration at each time point. Theelimination constant (ke, min-1) was obtained from the log-linearregression of the concentration (y) versus time (t). The half-life wascalculated as T½ (min)=0.693/ke. The microsomal intrinsic clearance(Clint, μL/min/mg of protein) was calculated by the following equation:

${{Clint}\mspace{11mu}\left( {\mu\;{{L/\min}/{mg}}\mspace{14mu}{of}\mspace{14mu}{protein}} \right)} = {\frac{V \times 0.693}{T\;{1/2}} = {V \times {Ke}}}$Where${V\left( {\mu\;{L/{mg}}} \right)} = \frac{{Volume}\mspace{14mu}{of}\mspace{14mu}{incubation}\mspace{11mu}\left( {\bullet\; L} \right)}{{Protein}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{incubation}\mspace{11mu}({mg})}$

Results are shown in Table 3.

TABLE 3 Microsomal stability Microsomal half life Clint uL/ stabilityassay -k (min) min/mg prot Cmpd 2 (HLM) 7.03E−04 ± 1.9E−05 986 ± 27 1.17± 0.03 Cmpd 2 (RLM) 1.26E−03 ± 1.9E−04 404 ± 28 6.19 ± 0.42 Loperamide3.25E−02 21 ± 1 54.07 ± 1.73  (HLM) Loperamide 7.53E−02  9 ± 0 270.99 ±2.98  (RLM)

Example 36 Animal Studies

All animal studies were approved by the Montreal Heart Institute (MHI)Animal Care and Ethical committee. Pharmacokinetic experiments wereperformed in collaboration at the Montreal Heart Institute incollaboration with the Platform of Biopharmacy at Universite deMontreal. Briefly, Compound 2 (formulation PG:H20; 50:50 stock solution2.5 mg/ml) was administered intravenously (iv) at 5 mg/kg (n=4) or byoral gavage (PO) at 25 mg/kg (n=4) in adult Sprague-Dawley male rats(˜200 g). Blood samples were collected at 5, 15, 30, 60, 120, 240, 360,480, 1440 min for which 125 μL of precipitation solution (80%acetonitrile and 20% methanol containing IS Loperamide at 0.1 μM) wereadded. Samples were mixed and centrifuged at 15000 rpm for 5 min. 50 μLof supernatant were transferred to an HPLC plate, and 100 μL ofwater+0.1% formic acid were added. A 16-point calibration curve withexample 2 was prepared in blank rat blood, ranging from 0.002 to 15 μM.Samples were analyzed in MRM mode using LC-MS/MS. The calibration curvewas plotted using the ratio of the analyte peak area and the IS peakarea, using a quadratic regression. Pharmacokinetic (PK) parameters werecalculated using Kinetica Software for PK/PD Data Analysis, Simulationand Reporting (Thermo Fisher), based on the blood concentrations of eachanimal. Results are described in Table 4 and illustrated in FIG. 20.

TABLE 4 Pharmacokinetic parameters Average StDev Parameter Unit (n = 4)(n = 4) Compound 2 (iv 5 mg/kg) AUClast μM*h 31.84 12.78 AUCtot μM*h31.90 12.83 MRT h 0.55 0.08 Lz 1/h 0.12 0.06 thalf h 6.60 2.67 Tmax h0.08 0.00 Cmax μMolar 81.12 24.53 Clearance mL/min 2.20 0.88 Vss L 0.080.04 Compound 2 (PO 25 mg/kg) AUClast μM*h 24.23 9.26 AUCtot μM*h 24.289.29 MRT h 3.44 1.42 Lz 1/h 0.24 0.02 thalf h 2.97 0.28 Tmax h 0.33 0.20Cmax μMolar 7.09 3.46 Clearance mL/min 13.44 6.26 Vss L 2.43 0.55 % F %15% 6%

For in vivo proof-of-concept animal studies, wild-type C57BL/6 male mice(˜25 g; obtained from Charles River Laboratories) or Golden Syrian (˜120g obtained from Envigo++) were housed in the 12 hour light/12 hour darkcycle, temperature and humidity controlled MHI animal facility.Following 5 days of acclimatization, animals were fed a chow or Westerndiet containing 48.5% w/w carbohydrate, 21.2% w/w fat, 17.3% w/w proteinand 0.2% w/w cholesterol (catalog no. TD.88137, Envigo) for 14 days.Example 1, 2, and 6 were mixed in either ethanol/propylene glycol/water;1:7:8 or 1:2:1 (v/v), or in phosphate-buffered saline (PBS; catalog no.311-010-CL, Wisent) containing 0.01% Tween 20 and administered by oralgavage at 5, 25 or 40 mg/kg/day for 3 or 10 days in mice maintained onchow or Western diet. Equivalent volume of vehicle containingcorresponding formulations were orally administered to mice daily anddesignated as placebo (vehicle). Plasma lipoprotein cholesterol profileswere obtained from 100 μL of pooled plasma injected on a Superose 610/300 GL (catalog no. 17-5172-01, GE Life Sciences) and eluted with PBSat a flow rate of 0.1 mL per min at 4° C. mounted on a AKTA explorersystem (GE Healthcare). Total cholesterol (catalog no. 439-17501, Wako)was quantified in total plasma and in each 0.3 mL collected fractions orin-line (Lipidomics Core of The Group on Molecular and Cell Biology ofLipids, University of Alberta) according to the manufacturer'sinstructions. Relative quantitation of AUC of total cholesterolcorresponding to non-HDL lipoprotein positive fractions obtained fromFPLC profiles are included in Table 3. Serum aspartate aminotransferase(AST) and alanine transaminase (ALT) were measured by the Montreal HeartInstitute biochemical clinical chemistry platform according tomanufacturer's recommendations. Results are described in Table 5.

TABLE 5 Efficacy and Safety of Compound 2 in hypercholesterolemic animalmodels LDL-C (%) Dosage Duration (FPLC TC) ALT AST Model n Diet (mpk/d)(days) (pooled plasma) (3 × ULN) (3 × ULN) C57BL/6 3 Chow 25 3 −31C57BL/6 4 HFD 40 10 −35 ± 2  0/4 0/4 C57BL/6 4 HFD 25 10 −28 ± 8 C57BL/6 4 HFD 5 10 −3 ± 8 Golden Syrian 3 HFD 40 10 −47 0/3 0/3 (n = 1)Golden Syrian 3 Chow 40 10 −12 Golden Syrian 3 HFD 40 10 −26 ± 12 GoldenSyrian 3 HFD 20 10 −40 ± 7  Golden Syrian 3 HFD 10 10 −15 ± 16 GoldenSyrian 3 HFD 5 10 −21 ± 10

Other Embodiments

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. A method of lowering low-density lipoprotein(LDL)-cholesterol level in the bloodstream of a subject, the methodcomprising administering to the subject a LDL-lowering amount of acompound having the formula:

wherein Y—X iS —C(R₇)(R_(7a))N(R′)C(O)—; —C(R₇)(R_(7a))OC(O)—;—C(R₇)(R_(7a))N(R′)C(R₆)(R_(6a))—; —C(R₇)(R_(7a))S(O)₂C(R₆)(R_(6a))—;—S(O)₂C(R₆)(R_(6a))—; —C(R₇)(R_(7a))C(R₆)(R_(6a))—; —O—C(R₆)(R_(6a))—;—N(R′)C(R₆)(R_(6a))—; or —C(R₇)(R_(7a))OC(R₆)(R_(6a))—, wherein each ofR₆, R_(6a), R₇, and R_(7a), is, independently, selected from H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle, R′ is selectedfrom H, C₁₋₆ alkyl, benzyl, S(O)₂R″, and C(O)R″, and R″ is selected fromC₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle; when Q isH, P and R₃ are absent; when Q is CH₃, P and R₃ are absent; Q is CH₂, orC(O); when P is H, R₃ is absent; P is —O—, —N(R₈)(R_(8a)), or is absent,wherein the R₈ and R_(8a) are selected independently from the groupconsisting of H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl andheterocycle; R₁ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆ alkene,halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branched alkyl),—C(O)(R₉), —C(O)O(R₉), —C(O)N(R₉)(R_(9a)), or —S(O)₂N(R₉)(R_(9a)),wherein R₉ and R_(9a) are selected independently from the groupconsisting of H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl andheterocycle; R₂ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆ alkene,halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branched alkyl),—C(O)(R₁₀), —C(O)O(R₁₀), or —C(O)N(R₁₀)(R_(10a)), wherein R₁₀ andR_(10a) are selected independently from the group consisting of H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle; or R₁ and R₂are ortho substituents that together form a carbocyclic or heterocyclicring system; R₃ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl,heterocycle, or is absent; R₅ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl,benzyl, phenyl, or heterocycle; or R₃ and R₅ combine to form aheterocyclic ring system; R₄ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, or heterocycle; and m is 0 or 1; or a pharmaceuticallyacceptable salt or solvate thereof.
 2. The method of claim 1, wherein Qis CH₂, P is —O—, R₃ is H, Y—X is —CH₂NHCH₂— or —CH₂NHC(O)—, R₁ isselected from a halogen, —OH or —OCH₃, R₂ is absent or selected from —OHand a halogen, R₄ is H or benzyl, and R₅ is H or benzyl, or apharmaceutically acceptable salt or solvate thereof.
 3. The method ofclaim 1, wherein Q is CH₂, P is —O—, R₃ is CH₃, Y—X is —CH₂OCH₂—, R₁ isselected from a halogen, —OH or —OCH₃, R₂ is absent or selected from —OHand a halogen, R₄ is H or benzyl, and R₅ is H or benzyl or apharmaceutically acceptable salt or solvate thereof.
 4. The method ofclaim 1, wherein the compound is5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 5. The method of claim 1, wherein the compound is5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 6. The method of claim 1, wherein the compound is5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 7. The method of claim 1, wherein the subject hasbeen diagnosed with atherosclerosis, hypercholesterolemia,hypertriglyceridemia, diabetic complications, dyslipidemia,hyperlipidemia, hypoalphalipoproteinemia, metabolic syndrome, stroke,vascular dimensia, chronic kidney disease, coronary heart disease,coronary artery disease, retinopathy, inflammation, thrombosis,peripheral vascular disease or congestive heart failure.
 8. The methodof claim 7, wherein the hypercholesterolemia is heterozygous familialhypercholesterolemia.
 9. The method of claim 7, wherein thehypercholesterolemia is homozygous familial hypercholesterolemia.
 10. Amethod of treating atherosclerosis, hypercholesterolemia,hypertriglyceridemia, diabetic complications, dyslipidemia,hyperlipidemia, hypoalphalipoproteinemia, metabolic syndrome, stroke,vascular dimensia, chronic kidney disease, coronary heart disease,coronary artery disease, retinopathy, inflammation, thrombosis,peripheral vascular disease or congestive heart failure, the methodcomprising administering to a subject in need thereof, a therapeuticallyeffective amount of a compound having the formula:

wherein Y—X iS —C(R₇)(R_(7a))N(R′)C(O)—; —C(R₇)(R_(7a))OC(O)—;—C(R₇)(R_(7a))N(R′) C(R₆)(R_(6a))—; —C(R₇)(R_(7a))S(O)₂C(R₆)(R_(6a))—;—S(O)₂C(R₆)(R_(6a))—; —C(R₇)(R_(7a))C(R₆)(R_(6a))—; —O—C(R₆)(R_(6a))—;—N(R′)C(R₆)(R_(6a))—; or —C(R₇)(R_(7a))OC(R₆)(R_(6a))—, wherein each ofR₆, R_(6a), R₇, and R_(7a), is, independently, selected from H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle, R′ is selectedfrom H, C₁₋₆ alkyl, benzyl, S(O)₂R″, and C(O)R″, and R″ is selected fromC₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle; when Q isH, P and R₃ are absent; when Q is CH₃, P and R₃ are absent; Q is CH₂, orC(O); when P is H, R₃ is absent; P is —O—, —N(R₈)(R_(8a)), or is absent,wherein R₈ and R_(8a) are selected independently from the groupconsisting of H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl andheterocycle; R₁ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆ alkene,halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branched alkyl),—C(O)(R₉), —C(O)O(R₉), —C(O)N(R₉)(R_(9a)), or —S(O)₂N(R₉)(R_(9a)),wherein R₉ and R_(9a) are selected independently from the groupconsisting of H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl andheterocycle; R₂ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆ alkene,halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branched alkyl),—C(O)(R₁₀), —C(O)O(R₁₀), or —C(O)N(R₁₀)(R_(10a)), wherein R₁₀ andR_(10a) are selected independently from the group consisting of H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle; or R₁ and R₂are ortho substituents that together form a carbocyclic or heterocyclicring system; R₃ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl,heterocycle, or is absent; R₅ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl,benzyl, phenyl, or heterocycle; or R₃ and R₅ combine to form aheterocyclic ring system; R₄ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, or heterocycle; and m is 0 or 1; or a pharmaceuticallyacceptable salt or solvate thereof.
 11. The method of claim 10, whereinQ is CH₂, P is —O—, R₃ is H, Y—X is —CH₂NHCH₂— or —CH₂NHC(O)—, R₁ isselected from a halogen, —OH or —OCH₃, R₂ is absent or selected from —OHand a halogen, R₄ is H or benzyl, and R₅ is H or benzyl, or apharmaceutically acceptable salt or solvate thereof.
 12. The method ofclaim 10, wherein Q is CH₂, P is —O—, R₃ is CH₃, Y—X is —CH₂OCH₂—, R₁ isselected from a halogen, —OH or —OCH₃, R₂ is absent or selected from —OHand a halogen, R₄ is H or benzyl, and R₅ is H or benzyl or apharmaceutically acceptable salt or solvate thereof.
 13. The method ofclaim 10, wherein the compound is5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 14. The method of claim 10, wherein the compound is5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 15. The method of claim 10, wherein the compound is5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 16. The method of claim 10, wherein thehypercholesterolemia is heterozygous familial hypercholesterolemia. 17.The method of claim 10, wherein the hypercholesterolemia is homozygousfamilial hypercholesterolemia.
 18. The method of claim 1 or 10, whereina pharmaceutical composition comprising the compound or the salt orsolvate thereof is administered to the subject.
 19. A method of inducinglow density lipoprotein receptor (LDLR) expression in a cell, the methodcomprising contacting the cell with an effective amount of a compoundhaving the formula:

wherein Y—X iS —C(R₇)(R_(7a))N(R′)C(O)—; —C(R₇)(R_(7a))OC(O)—;—C(R₇)(R_(7a))N(R′) C(R₆)(R_(6a))—; —C(R₇)(R_(7a))S(O)₂C(R₆)(R_(6a))—;—S(O)₂C(R₆)(R_(6a))—; —C(R₇)(R_(7a))C(R₆)(R_(6a))—; —O—C(R₆)(R_(6a))—;—N(R′)C(R₆)(R_(6a))—; or —C(R₇)(R_(7a))OC(R₆)(R_(6a))—, wherein each ofR₆, R_(6a), R₇, and R_(7a), is, independently, selected from H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle, R′ is selectedfrom H, C₁₋₆ alkyl, benzyl, S(O)₂R″, and C(O)R″, and R″ is selected fromC₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle; when Q isH, P and R₃ are absent; when Q is CH₃, P and R₃ are absent; Q is CH₂, orC(O); when P is H, R₃ is absent; P is —O—, —N(R₈)(R_(8a)), or is absent,wherein R₈ and R_(8a) are selected independently from the groupconsisting of H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl andheterocycle; R₁ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆ alkene,halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branched alkyl),—C(O)(R₉), —C(O)O(R₉), —C(O)N(R₉)(R_(9a)), or —S(O)₂N(R₉)(R_(9a)),wherein R₉ and R_(9a) are selected independently from the groupconsisting of H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl andheterocycle; R₂ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆ alkene,halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branched alkyl),—C(O)(R₁₀), —C(O)O(R₁₀), or —C(O)N(R₁₀)(R_(10a)), wherein R₁₀ andR_(10a) are selected independently from the group consisting of H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle; or R₁ and R₂are ortho substituents that together form a carbocyclic or heterocyclicring system; R₃ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl,heterocycle, or is absent; R₅ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl,benzyl, phenyl, or heterocycle; or R₃ and R₅ combine to form aheterocyclic ring system; R₄ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, or heterocycle; and m is 0 or 1; or a pharmaceuticallyacceptable salt or solvate thereof.
 20. The method of claim 19, whereinQ is CH₂, P is —O—, R₃ is H, Y—X is —CH₂NHCH₂— or —CH₂NHC(O)—, R₁ isselected from a halogen, —OH or —OCH₃, R₂ is absent or selected from —OHand a halogen, R₄ is H or benzyl, and R₅ is H or benzyl, or apharmaceutically acceptable salt or solvate thereof.
 21. The method ofclaim 19, wherein Q is CH₂, P is —O—, R₃ is CH₃, Y—X is —CH₂OCH₂—, R₁ isselected from a halogen, —OH or —OCH₃, R₂ is absent or selected from —OHand a halogen, R₄ is H or benzyl, and R₅ is H or benzyl or apharmaceutically acceptable salt or solvate thereof.
 22. The method ofclaim 19, wherein the compound is5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 23. The method of claim 19, wherein the compound is5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 24. The method of claim 19, wherein the compound is5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 25. The method of claim 19, wherein the cell is acardiovascular system cell.
 26. The method of claim 19, wherein the cellis in a subject.
 27. A method of modulating LDLR mRNA activity in acell, the method comprising contacting the cell with an effective amountof a compound having the formula:

wherein Y—X is —C(R₇)(R_(7a))N(R′)C(O)—; —C(R₇)(R_(7a))OC(O)—;—C(R₇)(R_(7a))N(R′)C(R₆)(R_(6a))—; —C(R₇)(R_(7a))S(O)₂C(R₆)(R_(6a))—;—S(O)₂C(R₆)(R_(6a))—; —C(R₇)(R_(7a))C(R₆)(R_(6a))—; —O—C(R₆)(R_(6a))—;—N(R′)C(R₆)(R_(6a))—; or —C(R₇)(R_(7a))OC(R₆)(R_(6a))—, wherein each ofR₆, R_(6a), R₇, and R_(7a), is, independently, selected from H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle, R′ is selectedfrom H, C₁₋₆ alkyl, benzyl, S(O)₂R″, and C(O)R″, and R″ is selected fromC₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle; when Q isH, P and R₃ are absent; when Q is CH₃, P and R₃ are absent; Q is CH₂, orC(O); when P is H, R₃ is absent; P is —O—, —N(R₈)(R_(8a)), or is absent,wherein the R₈ and R_(8a) are selected independently from the groupconsisting of H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl andheterocycle; R₁ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆ alkene,halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branched alkyl),—C(O)(R₉), —C(O)O(R₉), —C(O)N(R₉)(R_(9a)), or —S(O)₂N(R₉)(R_(9a)),wherein R₉ and R_(9a) are selected independently from the groupconsisting of H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl andheterocycle; R₂ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆ alkene,halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branched alkyl),—C(O)(R₁₀), —C(O)O(R₁₀), or —C(O)N(R₁₀)(R_(10a)), wherein R₁₀ andR_(10a) are selected independently from the group consisting of H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle; or R₁ and R₂are ortho substituents that together form a carbocyclic or heterocyclicring system; R₃ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl,heterocycle, or is absent; R₅ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl,benzyl, phenyl, or heterocycle; or R₃ and R₅ combine to form aheterocyclic ring system; R₄ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, or heterocycle; and m is 0 or 1; or a pharmaceuticallyacceptable salt or solvate thereof.
 28. The method of claim 27, whereinQ is CH₂, P is —O—, R₃ is H, Y—X is —CH₂NHCH₂— or —CH₂NHC(O)—, R₁ isselected from a halogen, —OH or —OCH₃, R₂ is absent or selected from —OHand a halogen, R₄ is H or benzyl, and R₅ is H or benzyl, or apharmaceutically acceptable salt or solvate thereof.
 29. The method ofclaim 27, wherein Q is CH₂, P is —O—, R₃ is CHs, Y—X is —CH₂OCH₂—, R₁ isselected from a halogen, —OH or —OCH₃, R₂ is absent or selected from —OHand a halogen, R₄ is H or benzyl, and R₅ is H or benzyl or apharmaceutically acceptable salt or solvate thereof.
 30. The method ofclaim 27, wherein the compound is5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 31. The method of claim 27, wherein the compound is5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 32. The method of claim 27, wherein the compound is5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 33. The method of any one of claims 4 to 6, 13 to 15,22 to 24, or 30 to 32, wherein the compound is administered incombination with a statin.
 34. The method of claim 33, wherein thestatin is atorvastatin.
 35. The method of claim 27, wherein themodulating comprises inducing LDLR mRNA activity.
 36. The method ofclaim 27, wherein the modulating comprises stabilizing LDLR mRNAactivity.
 37. The method of claim 1, further comprising administering asecond cholesterol lowering agent to the subject.
 38. The method ofclaim 37, wherein the second cholesterol lowering agent is a lipaseinhibitor, a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG CoA) reductaseinhibitor, a HMG CoA synthase inhibitor, a cholesterylester transferprotein (CETP) inhibitor, a bile acid absorption inhibitor, acholesterol absorption inhibitor, a cholesterol synthesis inhibitor, asqualene synthetase inhibitor, a squalene epoxidase or cyclase inhibitoror a combination of both, a microsomal triglyceride transfer protein(MTP)/Apolipoprotein B (ApoB) secretion inhibitor, a fibrate, niacinalone or in combination with lovastatin, an ion-exchange resin, anantioxidant, an acylcoenzyme A:cholesterol acyltransferase (ACAT)inhibitor and a bile acid sequestrant, an HMG-CoA reductase geneexpression inhibitor, an HMG-CoA synthase gene expression inhibitor, ora proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitor. 39.The method of claim 38, wherein the cholesterol synthesis inhibitor is astatin.
 40. The method of claim 39, wherein the statin is atorvastatin.41. The method of claim 1 or 10, wherein the subject, prior toadministration of the compound of Formula 1, was treated with a lipaseinhibitor, a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG CoA) reductaseinhibitor, a HMG CoA synthase inhibitor, a cholesterylester transferprotein (CETP) inhibitor, a bile acid absorption inhibitor, acholesterol absorption inhibitor, a cholesterol synthesis inhibitor, asqualene synthetase inhibitor, a squalene epoxidase or cyclase inhibitoror a combination of both, a microsomal triglyceride transfer protein(MTP)/Apolipoprotein B (ApoB) secretion inhibitor, a fibrate, niacinalone or in combination with lovastatin, an ion-exchange resin, anantioxidant, an acyl coenzyme A:cholesterol acyltransferase (ACAT)inhibitor and a bile acid sequestrant, an HMG-CoA reductase geneexpression inhibitor, an HMG-CoA synthase gene expression inhibitor, ora proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitor. 42.The method of claim 41, wherein the prior administration did noteffectively lower the cholesterol level in the bloodstream of thesubject.
 43. Use of a compound having the formula:

wherein Y—X is —C(R₇)(R_(7a))N(R′)C(O)—; —C(R₇)(R_(7a))OC(O)—;—C(R₇)(R_(7a))N(R′)C(R₆)(R_(6a))—; —C(R₇)(R_(7a))S(O)₂C(R₆)(R_(6a))—;—S(O)₂C(R₆)(R_(6a))—; —C(R₇)(R_(7a))C(R₆)(R_(6a))—; —O—C(R₆)(R_(6a))—;—N(R′)C(R₆)(R_(6a))—; or —C(R₇)(R_(7a))OC(R₆)(R_(6a))—, wherein each ofR₆, R_(6a), R₇, and R_(7a), is, independently, selected from H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle, R′ is selectedfrom H, C₁₋₆ alkyl, benzyl, S(O)₂R″, and C(O)R″, and R″ is selected fromC₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle; when Q isH, P and R₃ are absent; when Q is CH₃, P and R₃ are absent; Q is CH₂, orC(O); when P is H, R₃ is absent; P is —O—, —N(R₈)(R_(8a)), or is absent,wherein R₈ and R_(8a) are selected independently from the groupconsisting of H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl andheterocycle; R₁ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆ alkene,halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branched alkyl),—C(O)(R₉), —C(O)O(R₉), —C(O)N(R₉)(R_(9a)), or —S(O)₂N(R₉)(R_(9a)),wherein R₉ and R_(9a) are selected independently from the groupconsisting of H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl andheterocycle; R₂ is absent, C₁₋₆ alkyl, C₁₋₆ branched alkyl, C₁₋₆ alkene,halogen (F, Cl, Br, I), OH, —O—(C₁₋₆ alkyl), —O—(C₁₋₆ branched alkyl),—C(O)(R₁₀), —C(O)O(R₁₀), or —C(O)N(R₁₀)(R_(10a)), wherein R₁₀ andR_(10a) are selected independently from the group consisting of H, C₁₋₆alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl and heterocycle; or R₁ and R₂are ortho substituents that together form a carbocyclic or heterocyclicring system; R₃ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl, phenyl,heterocycle, or is absent; R₅ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl,benzyl, phenyl, or heterocycle; or R₃ and R₅ combine to form aheterocyclic ring system; R₄ is H, C₁₋₆ alkyl, C₁₋₆ fluoroalkyl, benzyl,phenyl, or heterocycle; and m is 0 or 1; or a pharmaceuticallyacceptable salt or solvate thereof, in the production of a medicamentfor lowering the cholesterol level in the bloodstream of a subject, fortreating atherosclerosis, hypercholesterolemia, hypertriglyceridemia,diabetic complications, dyslipidemia, hyperlipidemia,hypoalphalipoproteinemia, metabolic syndrome, stroke, vascular dimensia,chronic kidney disease, coronary heart disease, coronary artery disease,retinopathy, inflammation, thrombosis, peripheral vascular disease orcongestive heart failure, for inducing low density lipoprotein receptor(LDLR) expression in a cell or a tissue, or for inducing or stabilizingLDLR mRNA activity in a cell or a tissue.
 44. The use of claim 43,wherein the compound is5-[(4-fluoro-phenylamino)-methyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 45. The use of claim 43, wherein the compound is5-(4-fluoro-phenoxymethyl)-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.
 46. The use of claim 43, wherein the compound is5-[2-(4-fluoro-phenyl)-ethyl]-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylicacid hydroxyamide.